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Full text of "Vitamin content of meat"

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HARRY A. WAISMAN, Ph. D. 

Research Associate in Biochemistry 

University of Wisconsin 

and 

C. A. ELVEHJEM, Ph. D. 

Professor of Biochemistry 

University of Wisconsin 




Copyright 1941 

by 

Harry A. Waisman 



BURGESS PUBLISHING CO. - 426 SOUTH SIXTH STREET - MINNEAPOLIS, MINN. 





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I 



FOREWORD 

Man's debt to animals is very old. Long before he tilled crops he had 
acquired skill as a hunter, and the standard of living of our remote ancestors 
in most instances undoubtedly was rather directly proportional to the number 
and kind of animals they could kill. 

When animals were domesticated, enormous progress in human welfare was 
made possible. The Psalmist sang of "the cattle upon a thousand hills," and 
through the centuries of recorded history wealth has often and appropriately 
been measured by the livestock a man owned. 

In the complex industrial civilization that characterizes our twentieth 
century, most of this nation's population lives in cities and has little first- 
hand contact with agriculture. But the city man and his family that sees farm 
livestock only through the car windows during Sunday afternoon rides in the 
country, have much reason to be grateful to domestic animals. 

Not only do farm livestock supply large and critically important items 
in the human diet, as is technically explained in this book, but literally 
hundreds of thousands of our people are dependent for life itself upon the 
alchemy of domestic animals in transforming crude plant materials into bio- 
logical products like insulin and liver that are a daily necessity to persons 
afflicted with diabetes and pernicious anemia. 

The science of animal and human nutrition has made enormous progress in 
the last quarter century. Inadequate diets are now known to be the cause of 
many diseases, and methods have been developed to prevent and to cure these 
maladies. Research workers in the field of nutrition are proving marvelously 
efficient in saving lives and preventing ill health and suffering. When the 
knowledge of better diets 5s more widely distributed, and more closely followed, 
we can expect a much healthier and happier people. 

In a democracy like ours, it is particularly important that the general 
public be well informed regarding factors that influence the health and welfare 
of individual citizens. Under our system of government, it is not enough that 
we have knowledge and wise decisions by our chosen leaders. All our citizens 
must have the facts on which intelligent individual decisions can be made. 
This need for sound information is just as urgent in the case of nutrition as 
in economics or foreign affairs. 

The Agricultural Experiment Station of the University of Wisconsin has 
long emphasized the special importance of animal nutrition research in any 
program to advance agriculture as an industry and the welfare of farm people. 
We are gratified at the rapidly growing public interest in better nutrition. 
We hope this book will serve a useful part in helping students, governmental 
officials, and other persons in planning and bringing about the better nutri- 
tion of our nation' 3 population. 

Noble Clark 
Associate Director 
Agricultural Experiment Station 
University of Wisconsin 



1495,53 



PREFACE 



Hie aim of this book is to summarize the available information on the 
nutritive value of meat and meat products. It was the original intention of 
the authors to present this summary in the form of a "bulletin, but it soon 
became apparent that the amount of material wa3 too extensive for this form 
of publication. The fact that it is presented in book form should not leave 
the impression that there is any finality about the data. The entire subject 
is still in a dynamic stage and much more work needs to be done before defi- 
nitely reliable figures can be presented. We hope that the background of 
its preparation will be kept in mind when it is used. 



The literature has been reviewed in a critical manner and only those 
results which appear to be most reliable have been included. The work re- 
ported from our own laboratory has been in progress during the past five 
years and has been supported in part by grants from the National Live Stock 
and Meat Board through the National Eesearch Council. The work under these 
grants has been carried out by Br. Olaf Mickelsen, Mr. LaVell M. Henderson, 
and Mr. Junius M. Mclntire, as well as the authors. 

Individual chapters are devoted to the more important nutrients. Suf- 
ficient fundamental information is supplied in each case so that the value 
of meat in meeting the requirements of these nutrients can be considered in 
light of the most recent developments. The daily requirements listed for 
the individual vitamins and minerals are based on the values recently re- 
leased by the Committee on Food and Nutrition of the National Research Coun- 
cil. A summary chapter is included which gives general values for the 
vitamin content of the most important forms of meat products. 

We are especially indebted to Mrs. Earry A. Waisman for the long hours 
spent in helping to assemble this material and to Dr. D. M. Hegsted, Dr. A. 
E. Axelrod, and Mr. LaVell M. Henderson for reading parts of the manuscript. 



Harry A. Waisman 
C. A. El veh j em 



ii 



TABLE OF CONTENTS 



Foreword ... 

Preface 

Chapter I 

II 

III 

17 

V 

VI 

VII 

VIII 

IX 

X 

XI 

XII 

XIII 

XIV 

XV 



Page 
i 



ii 



Introduction - - 1 

The Nutritive Value of Meat _ 5 

Vi t amin A - 19 

Vitamin D _ 29 

Vitamin E and Vitamin K 37 

Vitamin C - k3 

Preparation of the Samples 55 

Proximate Analysis of the Animal Tissues 6l 

Thiamin (Vitamin Ej ) _ - 71 

Riboflavin 95 

Nicotinic Acid _ 121 

Pyridoxine (Vitamin Bg ) 151 

Pantothenic Acid - 167 

Additional Factors 193 

Meats as a Source of the Vitamin B 

Complex _ _ 202 



XVI Summary 209 



CHAPTER I 
INTRODUCTION 



I 
I 



CHAPTER I 

INTRODUCTION 

Early studies in nutrition were concerned largely with a qualitative estimate 
of the various factors in our food supply. Within recent years however ; fairly 
accurate methods have made possible a rather complete analysis of most food prod- 
ucts for a wide variety of nutrients . 

The kind of information available for different foods has followed very 
closely the evolution of our knowledge . The "beginnings of nutrition were related 
to the chance observations of a few physicians , chemists, and physiologists. As 
interest in this field increased there appeared the more orderly and systematic 
investigations on particular phases of the subject. It was logical for the early 
experimenters to feed certain foodstuffs to animals and watch the rate of growth 
or any manifestations of disease and ill health. The greatest progress was made, 
however, when the early workers introduced the white rat as a tool in biochemical 
research. The feeding of a particular food to experimental animals led the way 
but such experiments were difficult to interpret for several reasons . The protein 
level was adjusted so that optimum quantities of this constituent were fed, but no 
regard was given to the mineral content or to the other parts of the diet. Realiz- 
ing that the experiments were at fault from several viewpoints, the investigators 
next directed their efforts toward a better understanding of the value of proteins, 
fats, and carbohydrates. Attempts were made to raise rats and other animals on 
a mixture of grains, or upon a particular animal tissue containing equal quantities 
of protein and thus obtain information on the nutritive value of widely differing 
source materials. The early workers in the field of nutrition found that rats 
placed on diets containing meat grew very well, while other rats fed a combination 
of grains failed to develop normally. 

The extensive investigations on the role of proteins, fats, carbohydrates, 
and minerals in normal metabolism showed that the variations in the nutritive 
value of foods could not be explained solely by the difference in the amounts of 
these constituents and were next followed by a productive period, which dealt 
largely with the differentiation of the various food factors now called vitamins . 
The recognition that small amounts of these materials are necessary for normal 
growth, general well being and normal body function stimulated the research deal- 
ing with the concentration and isolation of the active materials from natural 
sources. Through the cooperative effort of the biochemist, bacteriologist, physio- 
logist and others, sufficient quantities of the pure materials were made available 
to the organic chemist so that the structure could be determined and synthesis of 
the vitamin accomplished. Large quantities of these compounds were thus possible 
for widespread use by the clinician and experimental investigator in a variety of 
deficiencies. 

As the knowledge of nutrition increased more recognition was given to the 
relation of the everyday diet to the maintenance of good health. Perhaps the most 
thorough studies on this subject have come from the work done by McCarrison and 
Aykroyd on the food and health habits of the peoples of India in the different 
sections of that country. From their observations covering many years these 
workers conclude that diet is of primary importance in the maintenance of health, 
in the perfection of physique, and in the vigorousness of youth and age. G.T. 
Wrench in his book, ; 'The Wheel of Health" points out the superiority of the in- 
habitants of the Native State of Hunza who consume considerable quantities of 



•1- 



vegetables, meat, and milk and explains how this superiority is related to their 
diet. The Sikhs and the Pathans are next in health and longevity "because they eat 
a somewhat similar diet hut which does contain more grains. The Najiris, Punjabs 
and Bengals are not as fortunate in their eating habits as the tribes already 
cited since they consume a less varied diet consisting mainly of rice and fish, 
and the incidence of disease in these tribes can be directly correlated to the 
deficient diet consumed. More attention is at present being given to the subject 
of diet in this country. Several workers are now becoming interested in the ade- 
quacy of the American diet and its relation to the prevalence of certain diseases, 
longevity, and sub-clinical complaints. 

The investigations of foods in previous years were necessarily limited by 
inadequate methods, or by a lack of methods. As each new method became available 
all groups of foods were examined for that particular constituent. It is now 
necessary, in the light of our present knowledge to intensively investigate each 
group of our foods . "While meat and meat products were studied mainly a3 sources 
of protein, phosphorus and iron, it is essential that we turn our attention to 
other substances furnished by animal tissues. 

A summary of the nutritive value of meat or any one of the group of foods is 
especially significant at this time when human requirements of these substances are 
being formulated by the Food and Nutrition Committee of the National Besearch 
Council acting as an agent of the government. It is also opportune to point out 
the importance of meat and meat products as sources of the accessory food factors 
and to review the recent advances in the knowledge of the distribution of the 
newer members of the vitamin B complex. While it is recognized that most mono- 
graphs or books on the vitamins are outdated the moment they are published, the 
authors believe that the data gathered together here can serve as a guide until 
newer methods cf assay or analysis are able to supplement the existing values . 

The knowledge of the vitamins in the water soluble 3 group has accumulated 
so rapidly within the past few years that little has been done on the distribution 
of these substances in many of our natural foodstuffs. It has now reached the 
stage at which it Is necessary to consolidate the advances made in the chemical 
work by making intensive investigations on the occurrence of these food factors. 
The isolation and synthesis of thiamin, riboflavin, pyridoxine and pantothenic 
acid has all taken place within the last six or seven years. The discovery that 
nicotinic acid is the pellagra preventing vitamin, and the importance of thiamin, 
pyridoxine, riboflavin, and pantothenic acid in the field of nutrition have also 
been the results of the most recent years. Choline, inositol, para-aminobenzoic 
acid, and other compounds also appear to be of physiological significance. The 
part that meat plays in supplying the well established factors as well as the more 
recently differentiated ones will become clear in the following chapters. 

The majority of the data to be reported in the following chapters have been 
accumulated at the University of Wisconsin in the Department of Biochemistry within 
the past five years . The work was initiated with experiments dealing with the 
distribution of vitamin B in animal tissues and was followed oj investigations 
concerned with the riboflavin, nicotinic acid, pantothenic acid, and pyridoxine 
content of meats. As the investigations progressed the development of improved 
methods of assay for a particular vitamin furnished the means by which earlier 
values were checked. Many of the assays to be reported dealing with the B group 
of vi tamins were confirmed by several methods . 



The data on the B group of vitamins in meat are given in full since certain 
portions of the experimental methods will be of interest to many investigators. 
Some of the procedures used in this study have not been reported heretofore or are 



modifications of previously described methods . It is thus advisable to describe 
them in detail. More than 150 samples of meat have been used in these assays and 
the proximate analyses of these animal tissues are given. The available literature 
on the vitamins other than the B group is also summarized. It should be pointed 
out that some of the data in the literature has been omitted since their accuracy 
can ncv be questioned because of faulty technic or to the lack of a suitable re- 
ference standards . 



\ 



CHAPTER II 

THE NUTRITIVE VALUE OF MEAT 

Page 
Role of Meat in the Human Dietary 5 

Digestibility of Meat 5 

Meat For Growth, Reproduction, and Lactation 6 

Meat in the Pathological Diet 6 

Pro t eins o f Meat 6 

Fats and Fatty Acids in Meat 8 

Carbohydrate in Meat 9 

Minerals in Meat 9 

Literature Cited 13 



CHAPTER II 

TEE NUTRITIVE VALUE OF MEAT 

Role of Meat in the Human Dietary 

The terms, meat and meat products include the flesh of all animals used for 
food such as "beef, veal, mutton, lamb, pork, poultry, and game, together with 
those portions of the carcass which can he utilized for food or food supplements, 
such as the liver, kidney, spleen, "brain, heart, pancreas, and thymus. The tre- 
mendous increase in the extent of the meat packing industry within the last 25 
years attests to the increased use of meat in the human dietary in this country. 
It has "been estimated (52) that the 1937 per capita consumption of meat was 12^ 
pounds and the 1938 figure was 128 pounds . Although these figures do not agree 
exactly with the more carefully obtained data of Stiebling and Phipard (76), it is 
apparent that meat plays an important role in the American diet. 

Stiebling and Phipard (76) found that the quantity of meat consumed in differ- 
ent parts of the country varied with the other foods consumed. They also observed 
that the individual consumption of meat increased -with increased money expended 
for food. If $2.50 to $3-12 per capita expenditure per week for food was taken 
as a good average, it was found that persons in the north Atlantic cities consumed 
13^ pounds of meat and meat products, cits'- dwellers in the east north central 
cities consumed a like amount, east south central 101 pounds, and Pacific 115 
pounds. The figure for the east south central area is understandably lower if one 
takes into consideration the characteristic eating habits of that section. A 
greater percentage of fish and other 3ea food is consumed by these inhabitants. 
This study also includes figures on the per capita consumption by white families 
for 3 months periods divided into the seasons of the year. For purposes of cal- 
culating consumption levels of meat and meat products, the figures for the east 
north central or north Atlantic states were considered as the most typical. If 
one adds the values for the four seasons, the yearly consumption of beef, veal, 
mutton, lamb, pork, miscellaneous meat products, and poultry can be calculated. 
The average individual in the north Atlantic states in all food expenditure levels 
consumes 5^- pounds of beef and veal, 11. 3 pounds of mutton and lamb, 22.9 pounds 
of pork (exclusive of bacon and salt pork), 13-9 pounds of miscellaneous meat 
products, and l6.2 pounds of poultry. 

Digestibility of Meat 

The high digestibility of meat and meat products has been well established. 
In recent studies, Pittman and her co-workers (36,^3,66) found that the average 
coefficients of digestibilitjr for the nitrogen in human subjects ranged from 79. 3 
to 34.8 percent for beef round with the average for all subjects at 82.5. When 
similar trials were made with beef liver the values were from 73-7 to 86.6 per- 
cent. The average digestibility was 82.1 percent which is not significantly dif- 
ferent from that of beef round. This verifies the earlier work of Mitchell and 
Beadles (55)- The work of these and other investigators has shown that meat is 
readily absorbed after digestion and leaves little residue in the intestine. 
Wells, Pomaranc, and Ivy (86) found that "tenderized ham" is not as well digested 
at the end of one hour as baked ham. No differences in digestibility were found 
in the meats when they were fed to depancreatized and gastrectomized dogs. 



•5- 



~^^fl 



Meat for Growth. Reproduction , and Lactation 

Much work has "been done on the value of meats for growth, reproduction, and 
lactation. Scheunert and Venus (70) found that when beef or sheep muscle was the 
sole source of protein in the diet of rats, normal growth and reproduction result- 
ed. Kelson and Swanson (57) compared the nutritive value of meat diets with stock 
diets and found that "ingestion of a high beef diet seems to induce unusually good 
growth in the albino rat, the birth of a large number of litters, and the success- 
ful rearing of particularly large and healthy appearing young rats." Nelson and 
Swanson (58) also studied the value of meats for hemoglobin production together 
with experiments on growth, reproduction, and lactation and found that beef muscle 
greatly increased the hemoglobin concentration in rats that were fed cereal mix- 
tures . Pork fed in addition to autoclaved yeast produced the highest level of 
hemoglobin. 

Using the rat as the experimental animal it was 3hown that fresh liver con- 
tains factors necessary for the normal functioning of the reproductive mechanism. 
Preliminary investigations with rats on the value of booked tissues have indicated 
that cooked liver is superior to casein for optimum lactation. Although results 
of this kind are satisfactory for demonstrating the value of one protein prepara- 
tion over another, it should be remembered that the vitamin content of these pre- 
parations will undoubtedly affect the results . As the knowledge of the vitamin 
content of meat increases such information will have to be taken into account. 
This will be especially true when protein from widely different sources, such as 
liver protein, egg albumin, and blood proteins are compared. 

Meat in the Pathological Diet 

There exists an extensive literature on the value of meat and meat products 
for the alleviation of anemias, hyperthyroidism, celiac disease, and other clinical 
conditions. The value of liver in treating pernicious anemia is now well estab- 
lished, and steady progress is being made on the isolation of the several compon- 
ents which are believed to be part of the extrinsic factor. 

There existed a common opinion that a high protein diet was detrimental to the 
normal functioning of the kidney, and that the disease nephritis was due largely 
to the excessive ingestion of protein. However, recent work (30,^7,51) lias given 
evidence to disprove this contention. Although much of the early work was primarily 
the result of rat experimentation, the use of smaller amounts of protein in our 
diets has been the advice of such eminent nutritionists and physiologists as 
Chittenden and Hinhede. The use of goodly amounts of meat in the diet has a dis- 
tinct advantage, however, as regards the ability of most animal tissues to furnish 
a large share of our need for vitamins as will be shown in the following chapters. 

There may be further value in eating meats aside from their protein, mineral 
and vitamin content. Eecent work by Page and his co-workers (6k) can perhaps be 
taken as a suggestion for the consumption of greater quantities of kidney in our 
diets. Page found a substance in kidney that has a profound effect in lowering 
the blood pressure, and he has named this substance renin after the renal organ. 
These workers assert that if this substance, renin, is kept out of the diet of 
both hypertensive dogs and rats, the blood pressure remains elevated. The feeding 
of fresh kidney or kidney extracts will lower the blood pressure to a normal level. 

Proteins of Meat 



Much of the available data on the protein content of meat was obtained in the 
early days of biochemistry and nutrition when the attempt was made to verify the 
assumption that muscular tissue of most animals was alike in composition. It was 



thought that the muscle of "beef was similar to the muscle of pork or lamb in its 
amino acid content. This assumption was verified "by the analyses of many workers. 
Atwater and Woods (3) listed the analyses on the composition of meats from many 
species and from various portions of the carcass. Fresh "beef "brisket for example, 
contained 15-8 percent protein as N x 6.25, chuck 19-2, flank 19-6, loin 19-0, 
sirloin steak 18.9, and beef round 20.9 percent. These are the averages of the 
minimum and maximum values of a great many samples. Organ tissue showed a simi- 
lar wide range of values. Beef heart contained 16.0 percent protein, kidney l6.6, 
liver 20. k f lungs, l6.0, and tongue 18.9. The values for lamb muscle were nearly 
identical with those of "beef. Leg of lamb contained 18.6 percent protein, breast 
and chuck 19. lj and hindquarter 19-6. The protein analyses of mutton tissue was 
different than that of beef and lamb. Mutton chuck and mutton flank analyzed 
Ik. 6 and 1^.3 respectively. The forequarter gave 15.6 percent and the hindquarter 
I0.7. Most of the analyses for mutton give lower protein values than the corres- 
ponding tissue of beef, and this may be explained by the fact that there is more 
fat present in the former samples . The pork samples also contain considerably 
more fat than those of beef and the protein analyses are correspondingly lowered. 
Fresh pork ham conta5.nedl5.7 percent protein. The loin samples showed l6 .h per- 
cent and the shoulder cuts 13-3 percent. The tenderloin samples showed a somewhat 
higher figure, 18.9. The organs of pork showed 11.7 for brain, 17.I for heart, 
15-5 for kidney, 21.3 for liver, and 11.9 for lung. 

The protein content of meat will vary from 15 to 20 percent, the higher 
values being more typical of beef and veal, while the lower values are usually for 
pork and mutton. The work of Powick and Hoagland (68), Wright (91), Wright and 
Forsyth (92), Hall and Emmett (18), Chatfield (9) among others furnish the basis 
for the above conclusions . 

The analyses for the individual amino acids of fresh meat, have shown that 
the animal proteins are biologically complete proteins. Several studies have been 
made on the amino acids of total meat proteins rather than on the individual muscle 
proteins because of the difficulties in preparing these in the pure state. These 
determinations have been summarized by Winton and Winton (90). The amino acid 
analyses of muscular tissues by Osborne and Jones (63) and the more recent analy- 
ses of muscle and organ tissue by Block and Boiling (k) are given in Table I. 

Clayton (10,11) gives evidence that muscle, liver, and kidney contain a good 
proportion of all the amino acids necessary for growth, maintenance, and reproduc- 
tion. The classical experiments of Eubner and Thomas have conclusively shown the 
high nutritive value of meat protein. Thomas (79) found that the biological value 
of meats was superior to the protein of other foodstuffs, and Starling (75) made 
the conclusion that animal protein is more economical than vegetable protein. A 
comparison of the amino acid content of muscle protein to that of the amino acids 
found in the milk protein, casein, shows that the distribution of the amino acids 
is approximately the same (56). 

One phase of protein investigations in meat has been the study of the specific 
dynamic action (69) of animal tissues. The value of certain tissues over others 
for producing this "extra heat" stimulation has been ascertained by several groups 
of workers. The effect of feeding meat on basal metabolism and upon respiratory 
metabolism following thyroidectomy as well as other conditions have been investi - 
gated (2,^5,56,87,88). Lusk, Williams, and Riche (k6) showed that when a quantity 
of meat was g:ven to a dog there was an increase in heat production above the 
caloric energy contained in that protein. This specific dynamic action of protein 
has been determined to be about 30 percent. In a very recent paper, Kriss (3^-) 
has critically reviewed the extensive researches of other workers and their 
theories of specific dynamic action. He has expressed his own results in ten dif- 
ferent ways so as to bear on the relationships between the heat increments and the 



8 

various metabolic factors. Kriss concludes that the dynamic effects of amino 
acids and proteins are ""by-products of intermediary chemical reactions and energy 
changes and they do not lend support to the idea that certain amino acids or cer- 
tain of their cleavage products act in the body as special metabolic stimulants 
in the pharmacodynamic sense." 

Fats and Fatty Acids in Meat 

The body fats of animals used for human consumption consist essentially of 
the mixed triglycerides of palmitic, stearic, and oleic acids, the proportions 
of these varying in the different species but remaining quite constant within the 
same species. Experiments on the elemental composition of body fat show that the 
average carbon content is usually 76.5 percent, hydrogen 12.0 percent, and oxygen 
11.5 percent (l). Animal fats are characterized by the "practical absence of 
glycerides of the water soluble, low carbon content acid3 ! ' (90). Milk fats con- 
tain glycerides of the lower acids of the aliphatic series in addition to the 
triglycerides found in body fats. Winton and Winton (90) claim that no volatile 
fatty acids or acids with higher than 18 atoms of carbon are present in hog, beef, 
or mutton fat, but all contain stearic, palmitic, oleic, and linoleic acids present 
as simple or mixed glycerides . These authors claim that alpha palmitodi stearin is 
characteristic of lard and that beta palmitodistearin is singular of beef and 
mutton tallow. The fat of beef, lamb, veal, and pork have been extensively inves- 
tigated, primarily from the point of view of the type of storage fat. 

The relationship of a nutritional dermatitis to the type of fat in the diet 
has proven to be of considerable importance. It has been shown by Burr and Burr 
(7) that linoleic acid is the fatty acid which will cure the scaliness of the skin 
and the faulty fur coat in rats that are maintained on a deficient diet. The com-, 
position of lard, beef tallow, and mutton tallow as quoted by Grossfeld (l6) indi- 
cates that the linoleic acid content of lard is 10.2 percent, 2.4 percent for beef 
tallow, and 7-3 percent for mutton tallow. From a nutritive importance, it is 
fairly obvious that these fats contain goodly 7- amounts of this particular food fac- 
tor. For those who are interested, Winton and Winton (90) summarize all this work 
in a chapter devoted entirely to animal fats . 

The tables on the composition of meat by Atwater and Woods (3) , as modified 
by Atwater and Bryant, again are able to furnish rather complete values for the 
fat content of many portions of the carcasses of veal, beef, lamb, and pork. Beef 
round contains 10.6 percent of fat in the fresh tissue. Such tissues as the 
brisket are higher in fat containing 28.5 percent, and top sirloin may also run as 
high as 43.7 percent of fat. The average values for the forequarter and hindquar- 
ter of beef are approximately the same, 18.4- percent. The organs of beef showed 
20.4 for beef heart, 4.8 for kidney, 4.5 for liver, and 3-2 percent for lungs. 
The veal samples were in the same range, but the mutton samples showed 36.8 percent 
fat for the chuck, 42.6 for the flank, and 17-5 for the hind leg. The pork samples 
were also quite high, and depending upon the particular cut analyzed the fat con- 
tent varied from 13.0 to 6l.O percent. Other fat analyses have been contributed 
~bj Powick and Hoagland (68), Wright (91), Wright and Forsyth (92), Hall and Emmett 
(18), and Chatfield (9). 

The work of Hilditch and his co-workers has been concerned with the component 
acids which occur in the depot fat of the ox and in plant material. An analysis 
for the higher saturated fatty acids, called "major constituents" by Hilditch, 
showed that of the total fat of beef tallow there was 38 to 40 percent of oleic 
acid, 26.5 to 31-0 percent of palmitic acid and 20.1 to 25.4 percent of stearic 
acid (23). Hilditch and Pedelty (24) gave the approximate determination of the 
unsaturated minor components of pig fats. The newer methods proposed decreased 



■WBBM 



9 

the time necessary for the analysis hut were not as quantitative as the longer 
procedures. The approximate determinations gave the percent -weight of pig fats 
as h2 to 50 percent for oleic acid and 5.0 to 8.0 percent for linoleic acid. The 
influence of the plane of nutrition on the composition and synthesis of the fat 
in the pig was studied "by Hilditch, Lea, and Pedelty (22). Among other conclu- 
sions, these authors claim that the quantity of linoleic acid in the "body fats 
is not more than and usually is less than half that available in the dietary fats. 
It is "believed that the unsaturated acids must he furnished in the diet since the 
analyses show that the amount deposited in the tissues is consistently less than 
that ingested. This "bears out the review of Burr (6) who made similar observa- 
tions on rats. Hilditch and Pedelty (25) have recently made comparisons between 
the perinephric fats and the outer hack fats from the same animal. 

Physical and chemical values on the various animal fats have "been determined. 
The changes in fat due to rancidity or acidity and the effect of antioxidants, 
catalysts, peroxides, and the influence of moisture on chemical composition has 
"been investigated to a considerable extent as shown by the summaries of Vinton and 
Vinton (90). The crystal structure of fat of the various species as well as the 
refractive index, the specific gravity, and the various fat constants have all 
been tabulated. 

Carbohydrate in Meat 

The carbohydrate content of meat has been considered negligible in comparison 
to the amount of protein and fat. In most analyses of meat the carbohydrate con- 
tent is expressed by the difference between moisture, protein, fat, and the total 
and is often called nitrogen-free extract. There are however, a few factors which 
influence the amount of carbohydrate that will be found in a muscle or an organ. 
The percent of sugar and glycogen in the tissues will vary according to the time 
that elapses between the slaughter and the analysis. A 50 percent loss of glyco- 
gen was observed by Trowbridge and Francis (83) in a beef muscle. In this sample, 
immediately after slaughter, there was a glycogen content of 0."1 percent, while 
after four hours storage the value was reduced to 0.32 percent. Another factor' 
which will influence the actual glycogen or sugar found in the meats is the meta- 
bolic condition at the time of slaughter. Still another consideration is the age 
of the animal. Trowbridge and Francis (83) found that beef muscle and beef liver 
from old animals contained more glycogen than those from young. The direct deter- 
mination of total carbohydrates is the best value that can be of practical use, 
and 3uch a determination by Toscani, Pupp, and McClellen (8l) gave for beef muscle 
I.36, tongue 1.09, liver 2.52, kidney O.J-t-0, and brain 1.08 percent. There is no 
favorable agreement between the available values in the literature. This is due 
largely to the various methods used, but the values give the impression that the 
liver is a richer source of glycogen than is muscle tissue (90). 

Minerals in Meat 

Meat and animal tissues in general furnish fair amounts of the mineral ele- 
ments. Early analyses were made by Katz (28) and Lawes and Gilbert (37,38) among 
many others. The analyses performed by Lawes and Gilbert were part of the study 
of the general composition of animal carcasses . Some analyses have contributed 
data which were obtained as part of analyses of the ash of certain tissues . Other 
values have been obtained as part of a larger project or for the direct comparison 
of the distribution of a particular element in several different tissues. The 
data on the occurrence of phosphorus, iron, and some of the minor mineral elements 
has been gathered together by "Winton and Winton (90). 



10 

Analyses for the minor inorganic elements lias received attention "because of 
the importance of these "trace" elements in "biochemical processes. It has been 
shown that zinc (26,77,80), copper (20), manganese (60), cobalt (50,61, 62,7^,85), 
magnesium (31, 35,^0,59), iodine (*l-8) together with iron are required by the mam- 
malian organism for normal body function.. 

The iron content of "beef muscle varies from about 25 milligrams to kO milli- 
grams per kilogram of fresh tissue. The liver, kidney, and spleen are usually 
higher than the muscular tissue, the values being 56 to 80 milligrams per kilo 
fresh organ. Several of the outstanding contributors on the analyses for iron 
have been Meyer and Eggert (53); Kenzui Kojima (29), Elvehjem and Peterson (12), 
Miller, Forbes, and Smythe (5*0, McHargue (50), Toscani and Reznikoff (82), and 
Forbes arid Swift (1*0. The available literature on iron has been summarized (90). 

The importance of manganese in the diet has stimulated the effort to analyze 
many of our important meat products for this element. Buttner and Miermeister 
(8), Peterson and Skinner (65), Skinner and Peterson (73), and McHargue (*t-9) have 
reported on the analyses for manganese. Ox liver contained 15 milligrams per 
kilogram of fresh material (*+9), while the lean muscle meat only contained a 
"trace". A beef T-bone steak contained O.65, lung 2.7, pancreas 3-3, veal chop 
1.3, pork chop 1.6, lamb chop 1.2, and calf brains 2.3, all expressed in milli- 
grams per kilogram of tissue (65). 

The work done on the determination of magnesium in animal tissues by Katz 
(28), Lawes and Gilbert (3S), and Gruzewska and P.oussel (17) ha3 furnished some 
of the more reliable analyses. The MgO content of most muscular tissues is ap- 
proximately 0.0^5 percent. Analyses on the entire carcass has given a range of 
0.032 to O.85 percent for ox, sheep, lamb, and hog. The magnesium content of the 
liver ash was 0.019 percent for the ox, 0.02 for the calf, and 0.021 for the hog 
(68). 

Only a few references are available on the existence of aluminum in animal 
tissues. Under hi 11, Peterm~n, Gross, and Krause (84) and Lehmann (39) have re- 
ported that aluminum is present in most tissues. The edible portion of beef showed 
5 milligrams per kilogram on the fresh basis, mutton h.3, pork h.h, calf liver 
17.3, pig liver 17-7 • Liver was again found to contain more of this element than 
the muscle tissue. 



One of the oustanding elements in" nutrition is c 
that it plays in certain anemias. Lindlow, Elvehjem, 
Nasyrova (32, 33), Hellwig and Quam (21), and McHargu 
the analysis for copper in the liver and muscular tis 
Ivanov (27) has also given some of the most recent co 
beef, and pork. The copper values for chicken ranged 
copper per kilogram of the dried muscle. Beef ran 51 
27 milligrams per kilo of the respective tissues. Th 
76 milligrams while the liver of sheep ranged from hj 
contained nearly the same as the liver of cattle. 



opper because of the role 

and Peterson (*!-2), Kogan and 
e (*t-9) have furnished some of 
sues of the various species. 
pper analysis of chicken, 
from 15 to 38 milligrams of 
.2 and pork ranged from 23 to 
e livers of cattle contained 
to 65 milligrams . Hog liver 



The zinc content of several animal tissues has been determined as the result 
of the interest shown in this element by virtue of its existence in the pancreas. 
Further interest in zinc has been stimulated by the work of the Wisconsin workers 
cited above, who showed its indispensable nature in the diet. Fisher and Scott 
(13), Sylvester and Hughes (78), Simakov (72), Ivanov (l8), and Lematte, Boinot, 
and Kahane (kl) have contributed some of the zinc analyses besides those given by 
the Wisconsin group. Muscle tissue contains 25 to 50 milligrams per kilogram on 
the fresh basis, while liver shows a range of 80 to 120 milligrams. McHargue (k-9) 



11 

has found lean ox neat to contain 15, ox liver 112, and the liver of a one week 
old calf 122.5 milligrams per kilogram. A bibliography of the heavy metals by 
Pope (67) includes a summary of the zinc content of foods and biologically impor- 
tant materials. 

A number of reports have dealt with the calcium content of meat. Burns (5), 
Williams (89), Katz (28), Laves and Gilbert (38), and Powlck and Hoagland (68) 
also give some values. It appears that the percentage of calcium in the muscle 
of the various species is about 0.018 expressed as CaO. The liver ash gave about 
0.006 percent of Ca. Meat cannot be considered a good source of calcium, and 
other foods must be relied upon to supply this element. 



TABLE I 
Amino Acid Content of Animal Tissues 





Ox Muscle 
(63) 


Heart 


Liver 
(h) 


Kidney 


Muscle 
(k) 


Glycine 


2.1$ 


■• 


- 


- 


- 


Alanine 


3-7 


- 


- 


- 


- 


Valine 


0.8 


3.2 


3.6 


3.8 


3.6 


Leucine 


11.7 


13. k 


11.6 


13.2 


11.0 


Serine 


- 


- 


- 


- 


- 


Cystine 


1.6 


1.1 


1.2 


l.k 


1.1 


Aspartic acid 


^•5 


- 


- 


- 


- 


Glutamic acid 


15.5 


- 


- 


- 


- 


Tyrosine 


2.2 


3.6 


3.0 


2.7 


3A 


Phenylalanine 


3.2 


5.3 


6.2 


5.5 


k.9 


Proline 


5.3 


- 


- 


- 


- 


Tryptophane 


1.3 


0.7 


1.0 


0.6 


0.6 


Arginine 


7-5 


6.8 


6.0 


7.2 


7.7 


Lysine 


7.6 


7.9 


k.6 


5.6 


6.9 


Histidine 


1.8 


2.3 


2.0 


1.8 


1.7 


Threonine 


- 


3.7 


k.6 


k.k 


3-5 


Isoleucine 


- 


k.o 


3-5 


3.5 


3.0 



13 
LITERATURE CITED 

1. Armsby, H.P. and Moulton, C.R. The Animal as a Converter of Matter and 
Energy. 1925. Chemical Catalogue Co., New York. 

2. .-Atkinson, H. V. and Luak, G. Further.- Experiments Relative to the Cause of 

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k. Block, R. J. and Boiling, D. The Determination of the Amino Acids, 19*1-0, 
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5. Burns, C. M. Calcium Content of Liver. Am. J. Physiol. 84, 39 (1935). 

6. Burr, G. 0. Animal Fats. Proc . Soc.Am. Prod., 32, 455 (1939). 

7. Burr, G. 0i and Burr, M. M. On the Nature and Role of the Fatty Acids 
Essential in Nutrition. J. Biol. Chem., 86, 587 (1930). 

8. Buttner, G. and Miermeister, A. The Manganese Content of Cow Milk, Beef and 
Some Other Foodstuffs. Zeit. Untersuch Lebensm., 65, 644 (1933)- 

9. Chat field, C. Proximate Composition of Beef. U.S. Dent. Agr. Dept. Circular 
389 (1926). 

10. Clayton, M. M. Comparative Value of Different Food Proteins for Reproduction 
and Lactation in the Rat, II, Milk, Eggs, and Meat , J. Nutr . , J5, 23 (1930). 

11. Clayton, M. M. The Comparative Value of Different Food Proteins for Repro- 
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12. Elvehjem, C. A. and Peterson, W. H. The Iron Content of Animal Tissues. 
J. Biol. Chem., 74, 433 (1927). 

13. Fisher, A. M. and Scott, D. A. Zinc Content of Bovine Pancreas. Biochem. J., 
29, 1055 (I929). 

Ik. Forbes, E. B.' and Swift, R.. W. The Iron Content of Meats. J. Biol. Chem., 
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15. Fox, H. M. and Ramage, H. Spectrographic Analysis of Animal Tissues. Nature, 
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Ik 

20. 



21, 



23 



2k 



25 



26 



28, 
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30. 

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32, 

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35. 



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Anemia. VII. Copper as a Supplement to Iron for Hemoglobin Building in the 
Rat. J. Biol. Chem., 77, 777 (1928) Ibid. XL, 797 (1928). 

Hellwig, A. H. and Quam, G. N. Copper Content of Beef and Hog Tissue. Food 
Ind., 2jkll (1930). 

Hilditch, T. P., Lea, C. H., and Pedelty, W. H. The Influence of High and 
Low Planes of Nutrition on the Composition and Synthesis of Fat in the Pig. 
Biochem. J., 33, '^93 (1939). 

Hilditch, T. P. and Longenecker, H. E. A Further Study of the Component Acids 
of Ox Depot Fat, with special reference to certain minor constituents . 
Biochem. J., 31, 1305 (1937). 

Hilditch, T. P. and Pedelty, W. H. Approximate Determination of Some of the 
Unsaturated Minor Components of Pig and Other Fats. Analyst., 6k, GkO (1939). 

Hilditch, T. P. and Pedelty, W. II. Body Fats of Pig. V. Component Glycerides 
of Perinephric and Outer Back Fat3 from Same Animal. Biochem. J., 3k, 9^1 
(19^0). 

Hove, E., Elvehjem, C. A., and Hart. E. B. The Physiology of Zinc in the 
Nutrition of the Pat. Am. J. Phys., 119 , 768 (1937). 

Ivanov, N.- Z. The Natural Copper and Zinc Contents of Meat and Other Foods. 
Voprosy Pitaniya, _7_, No. 2, Sk (1938). 

Katz, J. Die Mineral is ch en Bestandtheile des Muskelfleisches Pfluger's Arch, 
ges. Physiol., _§3, 1 (1896). 

Kenzul Kojima Iron in Normal and Pathological Tissues and Its Biological 
Significance. Nagoya J. Med. Sci.,_5, k? (1930). 

Keutmann, E. H. and McCann, W. S. Dietary Protein in Hemorrhagic Bright 's 
Disease. Effects upon the Course of the Disease with Special Reference to 
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Klein, H., Orent, E. R., and McCollum, E. V. The Effects of Magnesium Defi- 
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112 , 256 (1935). 

Kogan, A. M. and Nasyrova, K. M. Investigation of Food Products of Vegetable 
and Animal Origin for their Couper Content. Voprosy Pitaniya, kj No. 5, 30 
(1935). 

Kogan, A. M. and Nasyrova, K. M. The Copper Content of Foods. ,1V. The Copper 
Content of Animal Tissues, Eggs, and Leguminous Plants. Voprosy Pitaniya, 
U.S.S.R., Jj No. J+-5, 106, 116 (In English, ll6) •(1938) . 

Kriss ; M. The Specific Dynamic Effects of Amino Acids and Their Bearing on 
the Causes of Specific Dynamic Effects of Proteins. J. Nutr., _21, 257 (19^1). 

Kruse, H. D., Orent, E. P., and McCollum, E. V. Studies of Magnesium Deficiency 
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Chem., 96, 519 (1932). 



15 



36. Kunerth, B. L., Chitwood, I. M., and Pittman, M. S. Utilization of Meat by 
Human Subjects. III. The Utilization of Phosphorus and Nitrogen of Beef 
Heart. J. Nutr., 9, 685 (1935). 

37- Lawes, J. B. and Gilbert, J. H. Experimental Inquiry into the Composition 

of Some of the Animals Fed and Slaughtered as Human Food. Phil. Trans. Eoyal 

Society of London, p. kQ3 (1859). 

1. . 

33. Lawes, J. B. and Gilbert, J. H. Supplement to Former Paper Intitled "Experi- 
mental Inquiry into the Composition of Some of the Animals Fed and Slaughtered 
as Human Food" - Composition of the Ash of Entire Animals, and of Certain 
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39. Lehmann, K. B. The Determination of Aluminum and the Aluminum Content of 
Foodstuffs. Arch. Eyg., 106, 309 (1931). 

^0. Leroy, J. Necessiti Du Magnesium Pour La Croissance de la Souris Compt. Rend. 
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kj. Long, Z. and Pittman, M. S. Utilization of Meat by Human Subjects. II. The 
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kk. Lunde, G., Kringstad, H., and 01s en, A. Uber die Bestiramung des Lactoflavins 
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h6. Lusk, G., Williams, H. B., and Riche, J. A. Metabolism of the Dog Following 
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ho. McCollum, E. V., Orent-Keiles, E., and Day, H. G. Newer Knowledge of Nutri- 
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Nickel, Cobalt in Kentucky Blue Grass. Ind. & Eng. Chem., 19, 2Jk (1927). 



McLester, J. S. Nutrition and Diet in Health and Disease. 1939, P- 73 "77 
W. B. Saunders Co., Philadelphia. 



51. 

52. Meat and Live Stock Digest, 19, No. 6, Jan. 1939. 



16 
53- 

55. 



57. 



58.. 



59 



6o 
61. 

62, 

63. 
61+ 

65. 
66, 

67. 
68. 



Meyer, A. E. and Eggert, C. Iron and Copper in Liver and Liver Extracts. 
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Mitchell, H. H. and Beadles, J. R. The Protein Value in Nutrition of Beef 
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Mitchell, H. H. and Hamilton, T. S. The Biochemistry , of the Amino Acids. 
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17 

69. Rubner, M. Die Gesetze des Energie verbrauchs bei der Ernahrung. Leipzig, 
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11 
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18 

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36T (1927). 



CHAPTER III 
VITAMIN A 

Page 
Physiology and Pathology of Vitamin A 20 

Therapeutics of Vitamin A 20 

Methods of Vitamin A Assay 21 

Vitamin A in Animal Tissues 22 

Literature Cited 25 



CHAPTER III' 
VITAMIN A '•■:"■ 

One of the accessory food factors that received early recognition was vita- 
min A. It is still known as vitamin A for no official "body has given it' a chemical 
name. This does not imply however, that little is known about its chemistry, or 
its physiology, or even its pathology. On the contrary, it is the most thoroughly 
investigated food factor from these viewpoints. The enormous amount of work on 
the isolation and properties of vitamin A culminated in the elucidation of the 
structure and synthesis of the vitamin. This accomplishment has "been the reward 
of the combined efforts of the investigators in the field. The formulae for 
vitamin A and for y3 -carotene are given below: 



V 


V* 






/\ * 

H o C — C = 


H 


? H 3 E 
-6 = C 




I 







HgC 



C — CH_ 



H 



H 


CH 5 H H 


C- 


_c = c — C 




H 



OH 



Vitamin A C20H29OH 



Hr 



H 3 C x 



CH 



3 



h 3 C n 



■CH, 



H 2 C 



Hgt 



H„ 



H H CH 5 H H H CH 5 H H H H CH5 H H H CHj H H 

"c— c=c— c— c— c=c— c==c— c— c— c~ C — C^C— C=C — C=C — c' 



C— CH, 



fb -Carotene 



H^C— C 
3 \ 



C ^:-0 H 56 



C 



V CH, 



CH. 



Inspection of the two formulae shows that the fo ionone ring occurs in both fc caro- 
tene and vitamin A. One molecule of ft carotene can give two molecules of vitamin 
A upon halving the molecule. Other isomers of carotene such as the ocand X forms 
are only able to yield one molecule of vitamin A. The presence of the fb ionone 
ring and the unsaturated polyene side chain has been found to be essential for 
vitamin A activity. Not all carotenoids, therefore, have vitamin A activity, 
ralmer (50) lists some of the carotenoids, their important sources and their rela- 
tion to vitamin A. Through the use of the spectroscope, the existence of a second 
vitamin A, called vitamin A , has been postulated. Its existence is based on the 
absorption spectra of a compound other than vitamin A in the non saponifiable ex- 
tract of fish liver oil which reacts with antimony trichloride. The type of nomen- 
clature such as vitamin Ap should not be confused with the system of naming the 
various members of the B complex group of vitamins. In the latter each subscript 
denotes a separate vitamin, but here vitamin A2 only means another active form of 
vitamin A as yet unidentified. 



-19- 



20 

P'or a more complete review of the chemistry of vitamin A and related sub- 
stances, the reader is referred to the paper by Karrer and Wehrli (3^-) and to the 
monograph by Zechmeister (65). 

Physiology and Patholo gy of Vitamin A 

The role of vitamin A in the formation, development, and the normal activity 
of the epithelial tissues has been adequately shown in many clinical and experi- 
mental conditions. The various gradations of vitamin A deficiency are indicated 
by the microscopical changes in many organs and in a number of tissues. One of 
the most characteristic pathic changes in vitamin A deficiency is a keratinization 
of the epithelial tissue. This change may occur in the epithelial cells of the 
glands as well as in the stratified cells of the skin. 

In most animals, the eye i3 usually involved in vitamin A deficiency. Necro- 
sis of the cornea and metaplasia of the epithelium of the conjunctiva are seen in 
late stages of the avitaminosis . Visual processes are also affected by the absence 
of vitamin A since this substance has an intimate relationship with the normal 
mechanism for the production of visual purple. The teeth are affected as well as 
the respiratory tract. The atrophy of the respiratory mucosa precedes pneumonia 
in man. The skin is affected slightly in clinical patients deficient in vitamin 
A. There is a dryness with a mild exfoliation. In experiments with rats, hyper- 
keratosis of the stomach lining with ulcerations has been observed. Baumann and 
Steenbock (3) have shown that the changes in the vaginal epithelium could be used 
for the detection of a vitamin A deficiency. Coward (11) has also described the 
influence of vitamin A deficiency on the oestrus cycle of the rat. Complete ob- 
struction of the renal tubules and ureters of rats and chicks has been noted. 
Mellanby in 1931 ( J +l) and in 193^ ( J J-2) claimed that there was myelin sheath de- 
generation in the spinal cord of dogs maintained on vitamin A deficient diets. 
The polemic which ensued in the literature since Mellanby' s original thesis regard- 
ing myelin degeneration has now subsided to the extent where it can be said that 
there is "no substantial evidence that degeneration of myelin sheaths is a specific 
consequence of vitamin A deficiency." (k) . 

Therapeutics of Vitamin A 

Highly active concentrates of vitamin A obtained from fish liver oils as well 
as pure R> carotene are available for use in the clinic and in the laboratory. 
Natural vitamin A occurs as an ester and can be easily absorbed in the presence of 
fats. The fats are able to carry the yS> carotene across the intestinal wall. The 
administration of 10,000 to 20,000 international units (31) per day has given mark- 
ed results in cases of nightblindness . This condition has been considered as a 
subclinical vitamin A deficiency. For more advanced deficiencies higher dosages 
have also been advised. The use of vitamin A for cases of dry skin has been re- 
commended (23) . 



'Vitamin A has been used in treatment of certain cases of adolescent goiter 
(22), in hyperthyroidism (63), but further work is necessary to establish its 
therapeutic value in some of these conditions. Experimental urinary lithiasis has 
been produced in vitamin A deficiencies (10). The use of vitamin A in the treat- 
ment of burns, ulcers, and infections has been advocated in certain cases. Mc- 
Collum (39) first reported spontaneous infections in animals with xerophthalmia 
and several reports have given credence to the belief that resistance to infection 
is markedly decreased in man in vitamin A deficiency. 



21 

Since' # carotene and vitamin A may not be utilized to the same extent and 
since various factors may affect the absorption, it seems quite essential to dif- 
ferentiate between the utilization of vitamin A and fo carotene. Bile plays an 
important part in the utilization of P> carotene, but apparently has no effect on 
the utilization of vitamin A. Liquid petroleum causes considerable loss of ft 
carotene due to the solubility of the latter in the oil, yet it does not interfere 
markedly vith the absorption of vitamin A from the intestine (62,6k). The compo- 
sition of the diet other than the actual vitamin A supplied by the constituents 
has been reported to affect the absorption of vitamin A (7). With an understand- 
ing of these limitations as well as the recognition that the various methods will 
vary in accuracy and sensitivity, it is not surprising that the minimum vitamin 
A requirement for the prevention of night blindness is not too well established. 
From a number of papers which have appeared on the subject, it seems that 25 to 30 
international units per kilogram of body weight daily will prevent the night blind- 
ness (7). The results with the biophotometer, analyses of dietary surveys, and 
balance studies have all been taken into account in arriving at the human daily 
need for vitamin A. Booher (7) has considered the factors affecting vitamin A 
requirement, and with an allowance "for a fair margin of safety and for the main- 
tenance of a moderate storage of vitamin A in the body, a total of around 3,000 
units of vitamin A daily is suggested for the normal adult". The Technical Com- 
mission for the Study of Nutrition of the Health Organization of the League of 
Nations (37) recommends 2,000 to U,000 international units daily. The committee 
of Food and Nutrition of the National Research Council has placed the requirement 
of vitamin A at 5,000 international units. It appears from fairly reliable data 
that the vitamin A requirement of children is greater than that for adults. The 
recommended intake for pregnant and nursing women has been set at levels higher than 
that for men. Clausen has summarized the available literature on hypervitaminosis 
A and has discussed the effect of administration of high potency concentrates (9). 

Methods of Vitamin A Assay 

The tests available for the determination of vitamin A are of three kinds, 
biological, colorimetric and spectrograph! c or absorption methods. The official 
procedure for determining, vitamin A is by the rat growth method. The procedure is 
adequately described in the eleventh revision of the U. S. Pharmacopeia (6l). It 
is based upon the growth of depleted rat3 fed a supplement containing vitamin A. 
By a suitable comparison to the growth obtained with standard vitamin A, the actual 
quantity in the supplement is estimated. There are a number of necessary precau- 
tions in performing the assays. In order that reproducible results can be obtain- 
ed, the experimenter must make himself acquainted with the variables that are con- 
cerned. One other biologic method is the vaginal smear method, which depends upon 
the changes in the vaginal epithelium at various stages of abnormal oestrus during 
vitamin A deficiency (3,18,38). 

The color test with antimony trichloride has been used with only fair success. 
It is now used in some form of the original Carr -Price test (8) and depends upon ' 
the reaction of vitamin A with antimony trichloride in chloroform solution to give 
the typical blue color. The color is not entirely specific and is rather unstable. 
Various instruments have been used in reading the blue color thus giving rise to 
Lovibond blue units etc., but the modification of Dann and Evelyn (Ik) seems to be 
the mo3t promising. The use of suitable light filters eliminates interfering wave 
lengths of light so that reliable readings can be made. With this instrument the 
vitamin can be determined as fh carotene or as the blue color resulting from inter- 
action of the vitamin with antimony trichloride. French (2*0 has given data on 
the effect of various agents for controlling the fading of the blue color. He con- 
cluded that phosphoric acid was the most efficient reagent for this purpose but 
pointed out the limitations of the method when inactive chromogenic materials are 
present. Koehn and Sherman (35) have also discussed some of the limitations of the 
method , 



22 

The majority of the methods for the determination of vitamin A in. liver have 
"been modifications of the well established procedure of saponifying the liver tis- 
sue in alkali or in aqueous solution and followed by extraction with ether. The 
ether extract is then treated with the chloroform solution of the antimony tri- 
chloride reagent to give the "blue color. The complete procedure ha3 been describ- 
ed by Moore (kk,k5) . 

Vitamin A occurs in the liver primarily in the form. of vitamin A and not as 
lb carotene. The methods for determining [i carotene have been largely devoted to 
the estimation of that substance in plant materials. Guilbert (26) and Peterson, 
Hughes and Freeman (51) have offered a method for the determination of /3 carotene 
in forage. A recent contribution on the estimation of fi carotene in plant materi- 
als is that of Moore (*+3) . 

The spectrographs met" ods for the determination of vitamin A involves the 
measurement of absorption in that region of the spectrum where the vitamin A shows 
the maximum absorption. The concentrate of the oil or extract of the vitamin is 
dissolved in oil and is read in s, suitable instrument. There are a number of 
methods available for computing the results obtained from these measurements and 
they are summarized by Munsell (hj) . A recent review of the spectrophotometries 
method has been given by Ewing, Vandenbelt, Emmett and Bird (21). 

Vitamin A in Animal Tissues 

The most convenient form in which vitamin A may be supplied to the body is by 
fish oil concentrates, but by far the most common forms in which humans obtain 
their necessary quota of this factor are by liver, butter, milk, and eggs that 
they consume. Liver is considered the best source of vitamin A, whether it is 
from domestic animals or fish. There is a quantitative difference since fish oils 
are by far the richest source of vitamin A, 3ome species having as much as 7.5 
million international units per 100 grams of fresh liver (60). The content of 
vitamin A in fish liver oils varies widely depending on the source, the season 
of the catch, the refining, and storage processes (12). The vitamin A content of 
the fish body oils is definitely lower than the fish liver oils (6) . 

The available literature on the vitamin A content of meats is able to furnish 
only a few values. These are summarized in Table II. The values for beef liver 
show wide variation, the range being from 1000 international units per 100 grams 
to 16,000 international units. The various factors which influence the concentra- 
tion of the vitamin in the liver must be taken into account when the range of 
values is appraised. One sample of beef liver contained 9000 international units 
per 100 grams fresh weight according to one Investigator (46) who has tabulated 
some of the literature, while another group of workers (29) found 16,300 interna- 
tional units per 100 grams in another sample of beef liver. Calf liver samples 
have given values of 7000, 7500, and 3^,000 international units per 100 grams of 
the liver (56). Lamb liver showed a value of 3^000, and 26,500 international 
units per 100 grams. The results have been obtained from a number of workers who 
had access to samples from animals differing widely in their nutrition and environ- 
ment. Pork livers that were analyzed showed variations from 8000 to 17,000 inter- 
national units . 

It may be well to point out the influence of cooking on the vitamin A content 
of liver. One sample of sheep liver was analyzed before and after cooking, and it 
was found that 26,500 international units were contained in 100 grams of the liver 
before cooking, while after "cooking until tender", the sample contained 26,900 
international units per 100 grams (15). It is apparent that very little was lost 
by cooking, and the apparently slight increase in the vitamin content can be at- 
tributed to the probable loss of moisture in the cooked tissue before it was 
assayed . 



23 

Beef, veal, and lamb kidney shoved 1000, 1000, and 833 International units 
respectively. Lean "beef muscle contained 50 international units and pork muscle 
only showed a trace of the vitamin according to one group of workers (h-6) . Beef 
heart and lamb heart also were shown to contain traces of this vitamin. Some 
workers (53) have reported on the vitamin A content of the fat from beef, sheep, 
pork, and calf but have not given actual figures. Other investigators (l6) have 
claimed that mutton fat and pork fat were respectively 0.2 and 0.5 times the 
potency of butter fat. Such figures have little value. 

Guilbert and Hart (35) in California have demonstrated that the vitamin A 
content of beef liver from animals on pasture was higher than from those on a grain 
ration. They found that the livers of the former group contained 2080 internation- 
al units while the livers of the latter animals contained 1250 international units. 
McFarlane, Graham, and Richardson (ko) have observed that meat meal contains very 
little vitamin A. In another study, Guilbert, Miller, and Hughes (28) assayed the 
livers of pigs, sheep, and lambs and found that the range for pig liver was 75" 
300 international units and for sheep liver 10,000-16,000 international units 
while lamb liver contained 5000-13,200 international units per 100 grams of the 
tissue. 

Sen and Sharma (55) have investigated the livers of some Indian farm animals 
and have found that the liver of the bull varied from 128-256 international units 
per 100 grams. The goat liver was the highest of those investigated since they 
found 755"850 international units for this animal. The rabbit and calf livers 
were approximately in the same range showing a vitamin A content of 128 interna- 
tional units. These values are obviously too- low and do not compare to the more 
acceptable figures cited in the table. 

There are a number of papers in the literature which cannot be included in 
any table of quantitative figures. Many of these reports give comparisons between 
two tissues or merely contribute qualitative figures. For purposes of completion 
they are included in this discussion. Simonnet, Busson, and As3elin (57) found 
beef liver to be richer in vitamin A than the liver of guinea pig, rabbit, rat, 
and dog. 3ilek (5) found that vitamin A was present in the meat of the buffalo 
in amounts lower than in the cow. Von Euler and von Euler (19) found some little 
vitamin A in ox liver but did not give quantitative figures. Archer and Gill (l) 
found that lean meat of pigs contained very little vitamin A. Coward and Morgan 
(13) found no vitamin A in the calf liver they investigated. Schmidt -Nielsen, 
Flood, and Stene (5*0 have correlated the size of the liver with body weight and 
season, and have found that the vitamin A content varies with the food eaten, 
season, age, and sex. Houcamp, Sachsse, and Claus (30) have investigated the 
vitamin content of animal meal, meal meal, meat and bone meal, and found that some 
vitamin A is contained in these products, but that it is a poor source. These 
products were waste materials and contained much bone and were highly processed be- 
fore assay. The vitamin A content of the fat of prime beef has been investigated 
by Krizenecky (36), and from his study he concluded that roasting will destroy 
large quantities of the vitamin. Ender (.17 ) has claimed that in cattle the liver 
of the cow has a vitamin A reserve of approximately 5 times that present in the 
liver of the ox. Gyilbert and Hart (27) have found that chicken and turkey livers 
contain fair amounts of vitamin A. 

If it is assumed that the optimum daily requirements is 1+000 -5000 internation- 
al units of vitamin A for the normal adult, we can say that about 50 to 75 grams 
of fresh calf liver per day will supply adequate amounts of the vitamin. This is 
true since a perusal of the table indicates that the average vitamin A content of 
calf liver is approximately 7000 international units per 100 gram of fresh liver. 



2^ 

Since the requirement for vitamin A has been placed at 5000 international 
units on the baai3 of [b carotene, it would appear that when vitamin A ia supplied, 
less would he required. Vitamin A exists in liver primarily in that form, and 
very little is in the form of y5 carotene. There is evidently no loss in cooking 
as shown by the work of De and Majumdar (15), hut if there were as much as 30 
percent loss, a third of a pound of calf liver would still he able to supply the 
daily requirement. Although kidney contains less vitamin A than liver, it is 
decidedly higher than muscle tissue. It is then apparent that meats such as liver 
and kidney are able to supply a good share if not all of the daily requirement of 
this important vitamin. According to the study of Stiebling and Phipard (58) 
about one third of all the white families in the various income groups studied 
in their survey, obtained sufficient amounts of vitamin A. The best sources of 
this vitamin were the green leafy vegetables, butter, milk, and liver. The in- 
creased expenditure for food did not necessarily mean increased consumption of 
vitamin A, for the expensive foods did not contribute appreciable quantities of 
this vitamin. 

TABLE II 
Vitamin A Content of Animal Tissues 



Meat 


I.U. per 
100 gms. 


Eeference 


Beef Liver 


9,000 


k6 


Beef Liver 


16, 300 


29 


Beef Liver (on grain) 


1,250 


27 


Beef Liver (on pasture) 


2, 080 


27 


Calf Liver 


7,300 


56 


Calf Liver 


3^,000 


29 


Calf Liver 


7,000 


h6 


Pig Liver 


75-300 


28 


Pig Liver 


8,000 


52 


Hog Liver 


11, 700 


29 


Sheep Liver 


26,5V? 


15 


Cooked Sheep Liver 


26, 931 


15 


Lamb Liver 


15,000 


29 


Lamb Liver 


5,000-13,200 


28 


Sheep Liver 


10,000-16,000 


'.!5 ■ 


Turkey Liver 


162--250 


27 


Chicken Liver 


625-675 


27 


Beef Kidney 


1,000 


he 


Veal Kidney 


1,000 


ke 


Lamb Kidney 


833 


52 


Lean Beef 


50 


he 



25 
LITERATURE CITED 

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D'ept. Agr., Victoria, 2h, 60k (1926). 

2. Basu, N. K. Vitamin A and Fat Metabolism. Vitaminforschung, _6, 106 (1937). 

3- Baumann, C. A. and Steenbock, H. Vaginal Smear Method of Determining Vitamin 
A. Science, J6, kll (1932). 

k. Bessey, 0. A. and Wolbach, S. B. "Vitamin A Physiology and Pathology". 
The Vitamins, A Symposium, 1939. Am. Med. Assoc. 

5. Bilek, F. Fat Soluble Vitamins in Buffalo Milk. Proc. 11th World's Dairy 
Congress, Berlin, _3, 199 (1937). 

6. Bills, C. E. Anti-ricketic Substances. VI. The Distribution of Vitamin D 
with Some Notes on its Possible Origin. J. Biol. Chem. , 72, 751 (1927). 

7. Booher, L. A. "Vitamin A Requirements and Practical Recommendations for 
Vitamin A Intake". The Vitamins, A Symposium, 1939. Am. Med. Assoc. 

8. Carr, F. H. and Price, E. A. Color Reactions Attributed to Vitamin A. 
Biochem. J., 20, U97 (1926). 

9. Clausen, S. W. "The Pharmacology and Therapeutics of Vitamin A". The Vitamins, 
A Symposium, 1939. Am. Med. Assoc. 

10. Council on Pharmacy and Chemistry, Reports; Vitamin A and Urinary Lithiasis . 
J. Am. Med. Assoc, 105, 1983 (1935). 

11. Coward, K. H. Influence of Vitamin A Deficiency on the Oestrous Cycle of the 
Rat. J. Physiol., 67, 26 (1929). 

12. Coward, K. H., Dyer, F. J., Morton, P. A. and G-addum, J. H. The Determination 
of Vitamin A in Cod-Liver Oils. Biochem. J., 25, 1102 (1931). 

13. Coward, K. H. and Morgan, B. Quantitative Estimation of Vitamins A and D 
in Various Food Substances Cooked and Fresh. Brit. Med. J., 1935, II, 10^1. 

1^. Dann, W. J. and Evelyn, K. A. Determination of Vitamin A (Antimony Trichlor- 
ide). Biochem. J., J2_, 1008 (1938). 

15. De, N. K. and Majumdar, B. N. Further Studies on Factors Affecting the 
Vitamin A Activity of Animal and Vegetable Products. Ind. J. Med. Res., 25, 
357 (1938). 

16. Drummond, J. C. and Coward, K. H. Researches on the Fat-Soluble Accessory 
Substance. V. The Nutritive Value of Animal and Vegetable Oil and Fats Con- 
sidered in Relation to Their Colour. Biochem. J., Ik , 668 (1920). 

17. Ender, F. Difference in Vitamin A Contents of Cow and Ox Liver. Zeit. 
Vitaminf orschung, % 2^7 (193^) • 

18. Evans, H. M. and Bishop, K. S. On the Existence of a Hitherto Unrecognized 
Factor Essential for Reproduction. Science, _5_6, 650 (1922). 



26 

19. von Euler, B. and von Euler, H. Vitamin A in Animal Bodies. Arkiv. Kemi. 
Miner. Geol. 10B, No. 3, 6 pp. (1928). 

20. von Euler, H., Karrer, P., and Solmssen, IT. Homologe des Vitamins A (Axeroph- 
tols) und ein Abbauproducts des ^--Carotins, oc-apo -2 -carotinal. Helv. Chem. 
Acta, 21, 211 (1938). 

21. Ewing, D. T., Vandenbelt, J. M. , Emmet t, A. D., and Bird, 0. D. Spectro- 
photometry Determination of Vitamin A. Ind. Eng. Chem., Anal. Ed., 12, 639 
(19W). 

ii 

22. Fasold, H. Uber die Wirkung des Vitamin A auf das Ovar des Puberstatsalters . 
Klin. Wochnschr., _l6, 90 (1937). 

23. Frazier, C. N. and Ch'uan-K'uei, Hu Nature and Distribution According to Age 
of Cutaneous Manifestation of Vitamin A Deficiency. A Study of 207 Cases. 
Arch. Dermat. and Syph., 33, 825 (1936). 

2k. French, R. B. Assay of Vitamin A with the Photoelectric Colorimeter. Ind. 
Eng. Chem., Anal. Ed., 12, 551 (19*1-0). 

25. Green, H. N. and Mellanby, E. Vitamin A as an Anti -Infective Agent. Brit. 
Med. J., _2_, 691 (1928). 

26. Guilbert, H. R. Determination of Carotene as a Means of Estimating the Vita- 
min A Value of Forage. Ind. Eng. Chem.., Anal. Ed., _6, U52 (193*0. 

27. Guilbert, H. R. and Hart, G. H. Storage of Vitamin A in Cattle; Vitamin A 
Storage in Livers of Turkeys and Chickens. J. Nutr., _8, 2*3 (193*0 ', JL ^5 
(193*0. 

28. Guilbert, H. R., Miller, R. F., and Hughes, E. H. The Minimum Vitamin A and 
Carotene Requirement of Cattle, Sheep, and Swine. J. Nutr. 13, 5*1-3 (1937). 

29. Holmes, A. D., Tripp, F. and Satterfield, G. H. Beef, Hog, Calf, and Lamb 
Livers as Sources of Vitamin A. Food Research, 1_, hkj (193^). 

3C Koucamp, F., Sachsse, M., and Claus, G. Animal By-Products . II. Vitamin 

Content of Animal Meal, Meat Meal, Meat and Bone Meal, Bone Meal, Blood Meal, 
and Whole Meal. Zeit. Zucht. Riehe B. Tierzucht und Zuchtungsbiol., _25, 163 
-(1932). 

31. Jeghers, H. The Degree and Prevalence of Vitamin A Deficiency in Adults with 
a Note on its Experimental Production in Human Beings. J. Am. Med. Assoc, 
109, 756 (1937). 

32. Karrer, P. and Solmssen, U. fi -Carotinal, ein Abbauproduct des /5 -Carotins. 
Helv. Chim. Acta, _20, 682 (1937). 

33- Karrer, P., Solmssen, U., and Gugelmann, W. ,^-apo-^i-- carotinal, ein Wei teres 
Abbauproduct des $ Carotins . Helv. Chim. Acta, _20, 1020 (1937). 

3*+. Karrer, P. and Wehrli, H. 25 Jahre Vitamin A Forschung. Nova Acta Leopoldina 
N. S., 1, 175 (1933). 



27 

35. Koehn, C. J. and Sherman, W. C. The Determination of Vitamin A and Carotene 
with the Photoelectric Colorimeter. J. Biol. Chem., 152, 527 (19^0). 

36. Krizenecky, J. The Content of Vitamins A and D in Premier .jus Compared with 
that of Beef Tallow and Pork Lard, Chem. Listy, 3T, 88, 11^ (1937). 

37. League of Nations, Health Organization; Report on the Physiological Basis of 
Nutrition. Geneva, 1935- 

38. Macy, Icie G., Outhouse, J., Long, L. M., and Graham, A. Human Milk Studies 
Technique Employed in Vitamin Studies. J. Biol. Chem., 73, 153 (1927). 

39. McCollum, E. V. The Supplementary Dietary Relationships Among Our Natural 
Foodstuffs. J. Am. Med. Assoc, 68, 1379 (1917). 

kO. McFarlane, ¥. D., Graham, W. R., Jr., and Richardson, F. The Fat -Soluble 
Vitamin Requirements of the Chick. I. The Vitamin A and Vitamin D Content 
of Fi3h Meal and Meat Meal. Biochem. J. 25, 358 (1931). 

kl. Mellahby, E. The Experimental Production and Prevention of Degeneration in 
the Spinal Cord. Brain, 5k, 2^7 (1931) . 

k-2, Mellahby, E. Xerophthalmia, Trigeminal Degeneration and Vitamin A Deficiency. 
J. Path, and Bact., 38, 391 (193*0. 

^3. Moore, L. A. Determination of Carotene in Plant Material. Ind. Eng. Chem., 
Anal. Ed., 12, 726 (19^0). 

kk. Moore, T. Vitamin A and Carotene. V. The Absence of Liver Oil Vitamin A from 
Carotene. VI. The Conversion of Carotene to Vitamin A in Vivo. Biochem. J., 
2k, 692 (1930). 

^5. Moore, T. The Relative Minimum Doses of Vitamin A and Carotene. Biochem. J., 
27, 3Q8 (1933). 

h-6. Munsell, H. E. Vitamins and Their Occurrence in Foods. Milbank Memorial 
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h'J . Munsell, H. E. "Vitamin A Methods of Assay and Sources in Food". The Vita- 
mins, A Symposium, 1939. Am. Med. Assoc. 

48. Norris, E. R. and Church, A. E. A Study of the Antimony Trichloride Color 

Reaction for Vitamin A. II. The Dilution Curve of Cod Liver Oil with Antimony 
Trichloride Reagent. J. Biol. Chem., 87, 139 (1930); 35, ^-77 (1930). 

k-9 , Norris, E. R. and Danielson, I. 3. Comparison of Biological and Colorimetric 
Assays for Vitamin A as Applied to Fish Oils. J. Biol. Chem. _§3, i+69 (1929). 

50. Palmer, L. 3. "The Chemistry of Vitamin A and Substances Having a Vitamin A 
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51. Peterson, W. J., Hughes, J. S., and Freeman, H. F. Determination of Carotene 
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28 
53. 

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61+ . 



Rosenheim, 0. and Webster, T. A. The Sources of Supply of Vitamins A and D. 
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65. Zechmeister, L. Die Carotinoide, 193^. Berlin, Julius Springer, 



CHAPTER IV 

VITAMIN D 

Page 
Chemistry of Vitamin D 29 

Physiology and Pathology of Vitamin D Deficiency 30 

Therapeutics of Vitamin D 30 

Methods of Assay 32 

Vitamin D in Animal Tissues 32 

Literature Cited 35 



CHAPTER IV 

VITAMIN D 

Vitamin D prevents and cures rickets in infants and children and prevents 
softening of the "Dones in adults. This vitamin plays an important part in the 
proper utilization of calcium and phosphorus and therefore in the formation of 
good teeth and bones. Vitamin D is also necessary for the maintenance of normal 
tooth and bone structure. 

Chemistry of Vitamin D 

It is now widely accepted that there exists more than one compound with vitamin 
D activity. Several compounds have been shown to be important in the prevention 
cf rickets , but by far the two outstanding chemical substances are the irradiation 
products of ergosterol and 7-dehydro cholesterol. These compounds are definitely 
related in that the irradiation products have a somewhat similar chemical struc- 
ture. The formulae of these two active irradiation products are given here for 
purposes of comparison. 



HHHH Hm 
CH 5 -C-C=C-C-CC 




6h, n ch 5 



Bg 



HO 



-3 



HHHH H CH, 

CH.-C-C-C-C-CC 

"EH X CH 5 



/ s 




HO 



I 




Irradiated ergosterol 
or 
Calciferol (Vitamin D2) 



Irradiated 7-dehydro 
cholesterol 

or 
Vitamin D„ 



Activated ergosterol is usually called calciferol or vitamin D while activated 
7-dehydro cholesterol is named vitamin D3. The natural forms of vitamin D are 
not definitely known, and the terms Dg; &$> an< ^ ^k ara v& e & to denote physio- 
logically related compounds . There are numerous other but less important forms 
of vitamin D, and for those who are interested, Bills (l) has written a summary 
on this subject. 



-29- 



30 

Physiology an( l Pathology of Vitamin D Deficiency 

The physiology and pathology of vitamin D deficiency has been extensively re- 
viewed "by many authoritative workers. Complete monographs (28,29) have also con- 
tributed to an orderly presentation of the subject of rickets. So much has been 
done in the laboratory in an effort to understand the action of vitamin D in the 
body, that nearly all phases of biochemistry have played a part in the investiga- 
tions. The relationship between calcium and phosphorus in the blood, in the bone, 
and in the diet has been a constant challenge in obtaining information on the 
physiology of vitamin D. The recognition of the enzyme phosphatase has furnished 
further opportunity for study both in the bone and in the blood. Several workers 
have correlated the acid base content (.10,11,30) of the diet with the production 
of rickets, while Shohl and his collaborators (31 ) have dealt with body growth 
and development in various stages of the deficiency. That there is a relation- 
ship between the hormones, particularly that of the parathyroid, and vitamin D 
action is now well recognized. The role of the intestinal tract in the absorption 
of calcium and phosphorus both in the presence and in the absence of the vitamin 
is certainly an important one. There is the belief that higher acidities in the 
tract facilitate the absorption of calcium and phosphorus. The potential acidity 
of the food, which depends upon the acid forming ions, will also influence the 
absorption of calcium and phosphorus. There is then a balance which affects the 
actual retention of calcium and phosphorus in the body. The total effect is there- 
fore the resultant of the "true and potential acidity of the food" (28). Shohl 
(28) ha3 summarized the subject of the physiology of vitamin D action and pathol- 
ogy of vitamin D deficiency in a concise way. Thi3 author says that "the metabol- 
ism of vitamin D is closely related to the composition of the diet, especially 
the content of calcium and of phosphorus and the acid-base value. The metabolism 
of vitamin D is related to the parathyroid hormone, to phosphatase, and to other 
factors. The main action of vitamin D is to increase the absorption of calcium 
and phosphorus or to diminish their intestinal excretion. When the concentration 
of Ca " and (P0^) in the body fluid surrounding the degenerating cartilage cell, 
as measured by the state in the blood, is sufficiently great, deposition of salts 
in growing bone occurs." Much of the fundamental work on the physiology of vita- 
min D action has had practical application in the treatment of rickets, osteo- 
malacia, infantile tetany, and other related diseases. 

Therapeutics of Vitamin D 

It is generally agreed that the daily requirement of vitamin D for infants 
lies between 300 and ^00 International Units. The blood levels of calcium and 
phosphorus of infants fed this amount of vitamin are in the lower portion of the 
normal range. Higher dosages of vitamin D will permit levels of serum calcium 
and phosphorus to approach the top of the normal range (15 )• The diet of the in- 
fant will affect his requirement for vitamin D since there is evidence that in- 
fants fed cow's milk seem to require more vitamin D than breast fed infants. 
Infants fed human milk develop rickets significantly less than do those fed cow's 
milk (17). The need of vitamin D by the prematurely born infant is obviously not 
easy to determine, but it has been stated that this age group requires twice as 
much vitamin D as babies born at term, nearly 600 to 800 International Units. 

Jeans and Stearns (17) have expressed the belief that little evidence exists 
for the supposition that smaller amounts of vitamin D are necessary in older 
groups of children. The criteria for establishing the vitamin D requirement are 
the level of calcium and phosphorus in the blood, the gross appearance of the 
child at the time when the vitamin is given, the rate of growth of the child, and 
especially the utilization of calcium and phosphorus. Eecent work has shown that 



31 

the incidence of dental caries in younger children is decreased upon the adminis- 
tration of vitamin D (19, 21). The calcium and phosphorus retention can be stabi- 
lized by the administration of the vitamin as shown by a number of workers, most 
recently by Jeans and Stearns (16). 

Although it is agreed that the need for vitamin D decreases -with increasing 
age, it is with some justification that adolescent children have been said to re- 
quire the vitamin. Several studies have been concerned with the question of cal- 
cium retention on adequate and vitamin D low diets in adolescent boys and girls 
(12,33)« From the few reports available, it appears that vitamin D is essential 
for the retention of calcium in this age group and that the intake of the vitamin 
does not lower the need for calcium (13,33 )• 

The vitamin D requirement of adults will vary depending upon the period in 
which it is needed. One would expect that the amount of the vitamin necessary 
for men and non-pregnant women would be lower than that for pregnant and lactating 
women (9). Most investigators are agreed that an adequate vitamin D intake is 
advisable to promote normal calcification in the mother and in the foetus. The 
available data at present do not give precise dosages for administration during 
pregnancy and lactation,, but levels of 800 to 1000 International Units of vitamin 
D appear to be advisable with an abundant intake of calcium and phosphorus. While 
these quantities of the vitamin can serve as a guide, the part played by individual 
variation must be considered. It appears that with our present information no 
conclusions ca be drawn that are readily applicable to the majority of people. 

Vitamin D has been used therapeutically in a variety of clinical conditions. 
Some of these diseases are related to rickets, but the vitamin has been used for 
other complaints. Vitamin D has been used in several cases which are related in 
part to infantile rickets (23). The administration of a vitamin D containing oil 
has resulted in the alleviation of infantile tetany and in some cases of refrac- 
tory rickets (23). The vitamin has been used in the treatment of hay fever (2^), 
arthritis (8,3*0, psoriasis (2, 18) and other clinical entities. The rationale is 
not clear but some success has been reported in the treatment of these complaints. 

The Committee on Foods and Nutrition of the National Research Council has 
recommended that about lj-00 International Units of vitamin D per day should be the 
allowance of this vitamin for the various age groups in children and for pregnant 
and lactating women. The requirements for calcium for the development of normal 
bone structure will be dependent in part upon the size, age and activity of the 
individual child within the various age groups. The calcium allowance is more 
precise than the vitamin D requirement, being 1.0 gram for the infant and pre- 
school groups, 1.2 for the early school groups and l.k for the adolescent group. 
It is also recommended that about 1.5 to 2.0 grams of calcium 3hould be the amount 
ingested by the pregnant and lactating woman. The normal adult should obtain 0.8 . 
gram of calcium. 

The therapeutics of vitamin D brings up the qn.es tion of toxicity and excessive 
dosages of the vitamin. Reed (25) and others (3*0 have given very high dosages 
without any harmful effect. The tolerance level is evidently very high since 
dosages from 100,000 to 1,000,000 were administered with only mild nausea and 
frequent micturition. The danger of toxicity in children and infants is greater 
with these high levels than in adults. 



i 



32 

Methods of Assa y 

The production of rickets in the experimental rat furnished the basis for 
the observation that the healing of the induced rickets occurs in that area of 
the bony cartilage of the wrist bone known as the proliferative zone. This area 
between the shaft of the bone and the end of the bone is known as the metaphysis, 
and it is in this area that the calcification occurs when adequate amounts of 
calcium and phosphorus are supplied with a known amount of vitamin D. Within 
certain .limits the degree of healing has been shown to be proportional to the 
quantity of vitamin D that is administered (20). The degree of calcification is 
estimated by the width of the metaphysis that is uncalcified, and this line of 
demarcation between the calcified and uncalcified portion of the bone has given 
rise to the term "line test." Several modifications of the line test have been 
porposed, but the adoption of the U. S. Pharmacopeia method has unified the pro- 
cedures available for the estimation of vitamin D by means of this test. The 
U. S. Pharmacopeia unit of vitamin D was then adopted to coincide with the unit 
of measurement adopted by the Health Organization of the League of Nations. The 
International Unit is defined therefore as the "vitamin D activity of 1 milligram 
of the International standard solution of irradiated ergosterol, which has been 
found equal to that of 0.025 microgram of crystalline vitamin D." The official 
method provides for a comparison of the supplement under assay to that of a 
standard reference oil. The method has its limitations in that the production of 
rickets ia dependent upon the ingestion of a diet high in calcium and low in phos- 
phorus. If the supplement will disturb this ration, there will be a direct influ- 
ence on the healing process and thus on the vitamin D estimation. 

Another method that is of use in determining vitamin D potency is the bone 
ash te3t. This method is especially useful in the assay with chicks, although it 
can be successfully used with rat bones. The tentative A.O.A.C. method (32) in- 
volves the comparison of the bone ash of chicks receiving the vitamin D supplement 
with the bone ash of those bird3 which have received a definite amount of the 
standard reference oil. This method would serve as a good check on the line test 
were it not for the fact that chicks and rats respond differently to the different 
types of irradiation products which may be used as standards. 

Physical chemical methods are now being investigated as a means of differen- 
tiating between the various forms of vitamin D but these are not in the final 
stage of development. 

Vitamin D in Animal Tissues 

Table III summarizes the data available in the literature on the vitamin D 
content of animal tissues . The vitamin D content of beef and hog livers has been 
reported by Devaney and Munsell (7) to be between k0 and 50 International Units 
per 100 grams of the fresh livers. These workers also reported that lamb liver 
contained 20 International Units and calf liver 10 International Units per 100 
grams. Coffin (3) claimed that beef liver contained 9.0 International Units while 
beef steak contained 13.2 International Units per 100 grams of the fresh tissue. 
According to the work of Coward and Morgan {k ) the sample of calf liver which they 
analyzed contained no vitamin D. The work of Schmidt-Nielsen and Schmidt -Niels en 
(26,27) gives no quantitative figures, but it is claimed that the liver fats of 
elks, fowls, chick, white grouse, and auk are "deficient in vitamin D. " The liver 
of ox, calf, and whale showed a similar deficiency in this vitamin since these 
tissues contained less than 10 International Units of the vitamin. Munsell (22) 
has recently summarized some of the available data and has compared the values with 
fish muscle. It is readily seen from the table that salmon contains much more 
vitamin D than mammalian tissues, and the only other fairly good source of the 



33 

vitamin in domestic animals is the liver of the chicken. According to Hess and 
Weinstcck (1*0 the chicken liver they analyzed contained 67 International Units. 

Thi3 vide range of values for animal tissues is perhaps due in part to the 
fact that most of the figures have "been obtained indirectly as a result of those 
studies which have dealt with a subject distinct from vitamin D. The influence 
of various fats in the diets of the domestic animals was most usually reflected 
in the liver , and the majority of the determinations, therefore, were made on 
this organ of the body. The influence of various feeds on the deposition of vita- 
min D in the liver is also seen by the wide range of values. This influence is 
not limited to any one species for the samples of beef and calf liver show the 
same range of values. 

It can be concluded that beef and pork liver contain approximately h-5 Inter- 
national Units of vitamin D per 100 grams of fresh tissue. Calf liver has about 
10 International Units and lamb liver has nearly 20. Muscular tissues are perhaps 
only a fair source of vitamin D, since they contain approximately 10 International 
Units. Insufficient work has been done on the vitamin D content of animal tissues, 
but it seems clear that meat is not devoid of this vitamin. 



TABLE III 
Vitamin D Content of Animal Tissues 





Vitamin D (inter- 






national Units per 




Meat 


100 grams fresh wt. ) 


Reference 


Beef liver 


ii5 


22 


ti n 


^7 


7 


It 11 


9 


3 


Calf liver 


15 


22 


»t ti 





k 


n ii 


10 


7 


Pig liver 


^ 


22 


It 11 


hk 


7 


Lamb liver 


20 


7 


it »t 


17 


7 


Chicken liver 


50 


22 


tt w 


67 


5 


Steak, beef 


13-2 


3 


Salmon, Chum 


225 


22 


Chinook 


275 


22 


" Pink 


625 


22 


Red 


300 


22 


Liver fat, ox 






calf 


less than 10 


27 


pig 







35 



LITERATURE CITED 



1. Bills, C.E. "The Chemistry of Vitamin D" The Vitamins, a Symposium, American 
Medical Association (1939). 

2. Ceder, E.T. and Zen, L. Treatment of Psoriasis with Massive Doses of Crystal- 
line Vitamin D and Irradiated Ergosterol. A preliminary report, Public Health 
Report, 52, I5S0 (1937). 

3. Coffin, J. The Lack of Vitamin D in Common Foods. J. Am. Diet. Assoc, 11, 
119 (1935). 

k. Coward, K. H. and Morgan, B.G-.E. Quantitative Estimation of Vitamins A and D 
in Various Food Substances Cooked and Fresh. Briti Med. J., No. 3908, 10^1 
(1935). 



5. Daniel, E.P. and Munsell, H.E. Vitamin content of Foods. U.S. Dept of 
Agriculture, Miscellaneous Publication, 275 (1937). 

5. Davidson, L.T., Merrit, K.K. and Chipman, S.S. Prophylaxis of Rickets in 
Infants with Irradiated Evaporated Milk. Am. J. Dis. Children, 53, 1 (1937)- 

7. DeVaney, G-.M. and Munsell, H.E. Vitamin D Content of Calf, Beef, Lamb, and 
Hog Liver. J. Home Econ., ZJ, 2k0 (1935). 

3. Dreyer, I. and Reed, C.I. The Treatment of Arthritis with Massive Doses of 
Vitamin D. Arch. Phy. Therapy, 16, 557 (1935). 

9. Finola, G.C., Trump, R.A. and Gozelle, M. Bone Changes in the Fetus Follow- 
ing the Administration of Dicalcium Phosphate and Viosterol to the Pregnant 
Mother. Am. J. Ohst. and Gynec. , 3^, 955 (1937). 

10. Hamilton, B. and Dewar, M.M. Effect of Citrate and Tartrate on Experimental 
Rickets. Am. J. Dis. Children, 5^, 5^8 (1937). 

11. Hamilton, B. and Schwartz, C. Role of Acidosis and Alkalosis in the Etiology 
of Rickets. Am. J. Dis. Children, k6j 659 (1933). 

12. Henderson, J. M. and Kelly, F. C. Influence of Certain Dietary Supplements 
in Relation to the Calcium Requirements of Growing African Natives. J. Hyg., 
29, 1^29 (1930). 

13. Herbst, 0. Calcium und Phosphor beim Wachstum am Ende der Kindheit. Ztschr. 
f. Kinderh., Jj 1 ^ 1 (.1993 )• 

Ik. Hes3, A.F. and Weinstock, M. A Study of the Ant i -Rachitic Factor in Human 
and in Cow's Milk. Am. J. Dis. Children, jgj., t&5 (1927). 

15. Jeans, P.C. and Stearns, G. The Retention of Calcium by Infants Fed Evaporated 
Milk Containing Cod Liver Oil Concentrate. Proc. Soc. Exper. Biol, and Med., 
52, 1^58 (1935). 

16. Jean3, P.C. and Stearns, G. The Vitamin D Requirement of the Child, Proc. 
Am. Pediatric Soc, Am. J. Dis. Children, 5^-, 189 (1937). 

17. Jeans, P.C. and Stearns, G. "The Human Requirement of Vitamin D" The Vita- 
mins, a Symposium. Am. Med. Assoc (1939). 



36 

18. Krafka, J., Jr. Simple Treatment for Psoriasis. J. Lab. and Clin. Med., 
21, llk-J (1936). 

19. McBeath, E.C. Nutritional Control of Dental Caries. New York State J. Med., 
33, 1086 (1933). 

20. McCollum, E.V., Simmonds, N..., Shipley, P.G. and Park, E.A. Studies on Experi- 
mental Rickets. XVI. A Delicate Biological Test for Calcium- Depositing Sub- 
stances. J. Biol. Chem., 51, kl (1922). 

21. Mellanby, E. in "The Committee for Investigation of Dental Disease". The 
Influence of Diet on Caries in Children's Teeth (final report). Med. Res. 
Council, Special report series, No. 211, London, His Majesty's Stationery 
Office (1936). 

22. Munsell, H.E. Vitamins and Their Occurrence in Foods. Milbank Memorial Fund 
Quarterly, 1_8, 311 (19*K)). 

23. Park, E.A. "The Use of Vitamin D Preparations in the Prevention and Treatment 
of Disease" The Vitamin:;:, A Symposium. Am. Med. Assoc. (1939 )• 

2k. Rappaport, B.Z. ojid Reed, C.I. Viosterol of High Potency in Seasonal Hay 
Fever and Related Conditions. J. Am. Med. Assoc, 101, 105 (1933). 

25. Reed, C.I. Symptoms of Viosterol Overdosage in Human Subjects. J. Am. Med. 
Assoc, 102, I7II5 (193*0. 

26. Schmidt-Nielsen, S. and Schmidt-Nielsen, S. Vitamin D Deficiency of the 
Liver of Higher Animals. Kgl. Norske, Videnskab. Selskab. Forh.,_^, 190 
(1932). 

27. Schmidt-Nielsen, S. and Schmidt -Niels en, S. The Deficiency of Vitamin D in 
Mammals. Kgl. Norske Videnskab. Selskab. Forh., _5, 177 (1930). 

28. Shohl, A.T. "The Physiology and Pathology of Vitamin D" The Vitamins, A 
Symposium. Am. Med. Assoc (1939 )• 

29. Shohl, A.T. Mineral Metabolism. Rhelnho Id Publishing Co., New York (1939). 

30. Shohl, A.T. The Effect of the Acid-Base Content of the Diet upon the Produc- 
tion and Cure of Rickets with Snecial Reference to Citrates. J. Nutr., lk } 
69 (1937). 

31. Shohl, A.T., Brown, H.B., Chapman, E.E., Rose, C.3. and Saurwein, E.M. The 
Evaluation of the Phosphorus Deficiency of the Rickets Producing Diet. J. 
Nutr., 6, 271 (1933). 

32. Vitamin D Assay by Preventative Biological Test. J. Assoc. Off. Agr. Chem., 
20, 72 (1937). 

33. Wang, Chie Che, Kaucher, M. and Wing, M. Metabolism of Adolescent- Girls. 
IV. Mineral Metabolism. Am. J. Dis . Children, 52, kl (1936). 

,3c Wyatt, B.L., Hicks, R.A. and Thompson, H.E. Massive Dcses of Vitamin D in 
the Treatment of Proliferative Arthritis. Ann. Inter. Med., 10, 53*+ (1936). 



CHAPTER V 

VITAMIN E AND VITAMIN K 

Page 
Vitamin E 37 

Vitamin K 39 

Literature Cited kl 



S3 

Bi 



ti". 
of 
til 



CHAPTER V 
VITAMIN E AND VITAMIN K 

Two additional vitamins classed in the fat soluble group are vitamins E and 
K. Although the chemistry rjid physic logical action of these compounds have been 
described in detail by many investigators, there is insufficient data available on 
the distribution of these compounds in natural materials. However, for purposes 
of completion in this book, a short discussion will be given on each vitamin so 
that some basis for future investigations will be at hand. 

Vitamin E 

Vitamin E is the factor necessary in the diet which will insure the normal 
course of gestation in the rat. In rats maintained on a vitamin E deficient diet 
there occurs the death of the foetus followed by resorption of the embryo. If the 
animals are given vitamin E, chemically known as alpha tocopherol, succeeding 
gestations will be normal followed by the birth of healthy young (6). 

In certain groups of animals maintained on the vitamin E deficient diet it 
was observed that a type of paralysis occurred together with some nervous condi- 
tion (7). There was a striking similarity between some of the muscular dystro- 
phies of man and that seen in rats maintained on the vitamin E deficient diet. 
The occurrence of muscular dystrophy in rats was more pronounced when suboptimal 
amounts of the vitamin was given than when no vitamin was furnished in the diet. 
Goettsch and Pappenheimer also described nutritional muscular dystrophy in the 
guinea pig and rabbit (10 ). 

The attempt was soon made to apply these findings to humans "but at this time 
insufficient evidence prevents an appraisal of the therapeutic value of vitamin 
E. There have been reports from "both here and abroad which indicate that some 
cases of habitual abortion have responded to the administration of alpha toco- 
pherol or to wheat germ oil (2,20,21). A more practical demonstration of the 
effectiveness of vitamin E has been given by the work on muscular dystrophy. The 
evidence for the usefulness of the vitamin in the treatment of some dystrophies 
is accumulating rapidly. Several reports have pointed out the possible therapeu- 
tic failure with the vitamin. This is especially true in those clinical cases 
where permanent injury to the muscles might have taken place. The status of 
treating cases of amyotrophic lateral sclerosis is essentially the same as that 
of muscular dystrophy (23). 



Alpha tocopherol is one of the three tocopherols which have been isolated 
from natural sources. This compound also shows the greatest vitamin E activity. 
It is an oily liquid, and is soluble in most organic solvents and in oils. It is 
oxidized by ferric chloride, silver nitrate, gold chloride and other oxidizing 
agents. The formula and chemical name for vitamin E are given below: ' 



HO — 






n-rj . — -- \j 

ch! 



H H H 

CHp-CHp-CEp~C -CHp-CHp-CHp-C -CHo-CHp-CHp-C-CH^ 

CH, 



CH, 



CH, 



3 



y ch 5 ^ 

Vitamin E (alpha tocopherol) 
d-2,5,7,8-tetramethyl-2-(V,8 , ,12' trimethyl tridecyl)-6- 

hydroxy chromane 



■37- 



38 

Some studies have dealt with the relationship of vitamin E to the endocrine 
system, especially with the effect of the vitamin E deficiency on the reproductive 
organs and their normal production of the individual hormones. Drummond, Noble 
and Wright (k) found that the administration of vitamin E did not exert any 
effect on the ovaries, uterus, or vagina of adult hypophy3ectomized rats. The 
pituitary glands of E deficient male rats were found to contain an increased 
amount of gonadotropic hormone. The authors claimed that the effects of vitamin 
E deficiency are not produced by a hormonal imbalance as previously suggested by 
some workers . 

There exists a variety of conflicting evidence that neoplasms can be produced 
l>j feeding a crude ether extract of wheat germ oil. Mere experimental work to- 
gether with careful comparison of the particular conditions employed are necessary 
before the question can be settled satisfactorily. It has been claimed that a 
response in growth can be obtained in rat3 which have been maintained previously 
on vitamin E deficient diets (8). ; 

There is a dearth of information on the distribution of vitamin E. It is 
known to occur in plant materials especially in wheat and more specifically in the 
wheat germ oil. Butter fat contains some vitamin E (1*4-). Simmonds, Becker, and 
McCollum (13) have indicated that Certain samples of lard and cod liver oil con- 
tains considerable amounts of vitamin E as determined by rat bioas says. This is 
in contrast to the work reported by Evans and Burr (6). The vitamin E content of 
molasses, sorghum, and honey ha3 been reported by Taylor and Nelson (l7)> but no 
quantitative figures were given. Weatherby (22) has reported that 5 grams of 
avocado or 7-5 drops of the avocado oil was effective in producing cures in the 
female rats. The vitamin E content of soybean oil has been reported by Suzuki ' 
and his co-workers (15,16). An interesting investigations on the vitamin E con- 
tent of royal jelly by Evans, Emerson and Eckert (9) showed that no vitamin E 
activity was manifested when the jelly, its fat soluble fraction or the pollen 
from the comb was fed to female rats of proven sterility. 

The estimation of tocopherol in animal organs wa3 carried out by Karrer, 
Jaeger and Keller (12) by means of a gold chloride potentiometric titration 
method and by a color imetric method. Good agreement of the results was obtained 
with both methods after taking into account the error due to the carotene which 
occurred in some of the liver extracts. The potentiometric method gave 5*3 milli- 
grams per kilogram of fresh horse muscle, ^.9 for horse heart, I3.3 for horse 
liver, 6.25 for horse kidney. Cattle muscle showed 5.85 milligrams per kilogram 
and 9-5^ milligrams for cattle liver. A sample of hog lard gave approximately 
2.0 milligrams of alpha tocopherol per kilogram. As more refined methods become 
available further determinations for vitamin E will' undoubtedly show that more of 
the vitamin occurs in the organs having a higher fat content. Liver is an out- 
standing animal source of the vitamin. 

An investigation by several Iowa workers (19) has indicated that muscle tis- 
sue from goats maintained on vitamin E deficient diets contained insufficient 
vitamin E as indicated by the typical resorption gestations in avitaminosis E 
sterile rats which were fed the goat tissues. Although the goats were maintained 
on the vitamin E deficient diets, there was normal reproduction for several gener- 
ations. This demonstrates that goats do not, seem to require vitamin E, and that 
although the vitamin E content of the muscle and organs is nearly devoid of the 
vitamin normal reproduction is possible. 



39 



Vitamin K 



The existence of vitamin K was first postulated by Dam (3) in 1935 "when he 
noticed a "bleeding tendency in chicles maintained on a fat low diet. Dam noted 
that the "bleeding could "be controlled by the administration of certain oils, 
alfalfa, and the non-saponifiable non-sterol fraction of hog liver fat. He called 
the factor contained in these supplements the "koagulation vitamin" or vitamin 
K since it aided coagulation in some way. This vitamin is essential to insure 
the formation of a substance necessary for the clotting of blood within the nor- 
mal time limit, thus preventing hemorrhage. 

Method of assay for this factor were developed and contributed in a large 
measure to the rapidity with which the chemical isolation of the vitamin was 
accomplished. This was quickly followed by the determination of structure and 
synthesis of the active compounds. At present there are many compounds having 
vitamin K activity, but the names K^ and K/s have been given to the two naturally 
occurring substances. Both compounds have the l,k naphthoquinone nucleus, but 
it has been shown that the compound having a methyl group substituted in the 2 
position of the naphthoquinone is more active than the naturally occurring vita- 
min K-j_ and K^. These vitamins are stable to light, reducing agents, and heat, 
but are readily destroyed by alkali in alcoholic solution. Strong acids also 
destroy the vitamins. The formulae for vitamin Kj_ and 2 -methyl-1, ^-naphthoquinone 
are given below: 



HO , , x 

n A Vitamin K (2-methyi-3-phytyl-l, ^-naphthoquinone ) 



HC 



EC 



/ \ / \ 



C-CH 



/ \J 



3 



H 
C-CH-.-C: 



H 2 - 







H 



H 



/ 



CH, 



CH -CH -CH -C-CH -CH -CPI -C-CE -CH^-CH^-CH ° 



CH, 



j 1 wx^ wx 2 .^-w-vxig-w.ug. 

CH* CH. 



d \z 



3 



2 -methyl- 1, ^--naphthoquinone 



HC 



HC 



H 
-ft C 




C-CH- 



\ 



C v CH 



c 

II 




Vitamin K has received attention in the clinic by its marked action in pre- 
venting bleeding tendencies after surgery (5); in raising the prothrombin level 
of the blood (l), in obstetrical conditions of uterine bleeding and hemorrhagic 
disease cf the newborn (11 ), in complications due to obstruction of the bile duct 
and jaundice (18) and in several miscellaneous conditions. Vitamin K does not 
seem to be effective in cases of hemophilia.. There is evidence that man obtains 
a portion of his vitamin K requirement from the bacterial action in the gastro- 
intestinal tract. In cases where faulty absorption interferes with the normal 
utilization of vitamin K in the diet, bile salts have corrected the abnormal- 
condition. 



ko 



The distribution of vitamin K has "been studied only in a general way. It 
has always been claimed that green leafy materials contain large amounts of vita- 
min K. The original studies by Dam (3) indicated that hemp seed and certain vege- 
tables are good sources while yellow corn, unpolished rice and sunflower seeds 
are very poor. The hen's egg contains some vitamin K but is confined to the yolk 
portion. By far the richest source is hog liver. Calf thymus, ox kidney and ox 
adrenals contain only fair amounts of the vitamin. It has been found that calf 
brain, ox lung and ox muscle are poor sources of the vitamin. As yet no compre- 
hensive assays have been made on the vitamin K content of animal tissues, but it 
is apparent that those tissues which have a higher fat content will contain more 
vitamin K. 



in 

LITERATURE CITED 

1. Cheney, G. The Plasma Coagulation Time as a Simple Test for Vitamin K 
Deficiency. Am. J. Med. Sci., 200 , 327 (19^0). 

2. Currie, D.W. Vitamins for Habitual Abortion. Brit. Med. J., JL, 752 (1936). 

3. Dam, E. The .Ant i -hemorrhagic Vitamin of the Chick. Biochem. J., 29, 1273> 
(1935). 

k. Drummond, J.C., Noble, R.L. and Wright, M.D. Studies on the Relationship of 
Vitamin E to the Endocrine System. J. Endocrinology, 1, 275 (1939)' 

5. Editorial, Biliary Tract Surgery and the Bad Risk Case. Surgery, 7, 92^ 
(19^0). ~ 

5. Evans, H.M. and Burr, G.O. The Antisterility Vitamin Fat Soluble E. Memoirs 
of the Univ. of California, _8, I-I76 (1927 ). 

7. Evans, H.M. and Burr, CO. Development of Paralysis in the Suckling Young of 
Mothers Deprived of Vitamin E. J. Biol. Chem., 65, 273 (1928). 

8. Evans, H.M. , Emerson, G-.A. and Emerson, O.H. Growth Stimulating Action of 
Alpha -tocopherol. Proc. Soc. Exper. Biol, and Med. , 38, 197 (1938). 

9. Evans, H.M. , Emerson, G.A. and Emerson, O.H. Alleged Vitamin E Content in 
Royal Jelly. J. Econ. Entomology, 30, 6k2 (1937). 

10. Goettsch, M. and Pappenheimer, A.M. Nutritional Muscular Dystrophy in the 
Guinea Pig and Rabbit. J. Exp. Med., 5h, 1^5 (1931). 

11. Javert, C.T. Intra Uterine Onset of Hemorrhagic Disease of the Newborn. 
Am. J. Obst. and Gynec, kO, U53 (19^0). 

12. Karrer, P., Jaeger, W. and Keller, H. Estimation of Tocopherol in Animal 
Organs. Helv. Chim Acta, 23, k6k (l9*J-0); C.A. 30:5^. 

13. Simmcnds, IT., Becker, J.E. and McCollum, E.V. The Distribution of Vitamin E. 
J. Nutr., 1, 39 (1928). 

ih. Sure, B. Dietary Requirements for Reproduction. XI. The Potency of Butter 
Eat in Vitamin E. J. Biol. Chem., jk, 71 (1927). 

15. Suzuki, V., Waro, N. and Yoshikazu, S. The Occurrence of Vitamin E in Soy- 
bean Oil. Sci. Papers Inst. Physic. Chem. Res . , _23, 270 (193*0- 

16. Suzuki, V., Waro, N. and Yoshikazu, S. Further Evidence for the Occurrence of 
Vitamin E in Soybean Oil. Sci. Papers Inst. Physic. Chem. Res., 2k , 283 (193*0' 

17- Taylor, M.W. and Nelson, V.E. Molasses, Sorghum, and Honey as Sources of 
Vitamin E. Proc. Soc. Exp. Biol, and Med., J26, 521 (1929). 

18. Tovnsend, S.R. and Mills, E.S. Hemorrhagic Tendency Associated with Pro- 
thrombin Deficiency and its Treatment with Vitamin K and Bile. Canadian Med. 
Assoc. J., k2, 5^1 (19^0). 



k2 

19. Underbjerg, G.K.L., Thomas, B.N. and Cannon, C.Y. Effect of Decreased Body- 
Reserves of Vitamin E on the Reproduction of Goats, Proc. Amer. Soc. Animal 
Prod., p. 62 (1938). 

20. Vogt-Mueller, P. Die Behandlung des Habitue lien aborts mit Weizehkeimol. 
Klin. Wochenschr., 15, 1883 (1936). 

21. Wat3on, E. M. and Tew, M.P. "Wheat Germ Oil (Vitamin E) Therapy in Obstetrics. 
Am. J. Obat. and Gynec, 31, 352 (1936). 

22. Weatherby, L. Vitamins C, D, E (in avocados). Calif. Avocado Assoc. Year- 
book, p. 100-105 (1930). 

23- Wechsler, I.S. Recovery in Amyotrophic Lateral Sclerosis Treated with Toco- 
pherols (Vitamin E); preliminary report. J. Amer. Med. Assoc, lljf, 9^-8 
(19^0). 



CHAPTER VI 
VITAMIN C 

Page 
Chemistry and Physiology of Vitamin C kj 

Pathology of Vitamin C kk 

Vitamin C Therapeutics kk 

Methods of Assay for Vitamin C kk 

Vitamin C In Animal Tissues !f5 

Literature Cited ^9 



CHAPTER VI 



VITAMIN C 



Chemistry and Physiology of Vitamin C 

Adequate presentations of the chemistry of vitamin C including its identifi- 
cation, isolation, characterization, and synthesis have "been given "by King (27; 
28). The structure of the compound is closely related to that of the carbohydrat- 
es. The proposed syntheses of the vitamin use 1-xylose or d-glucose as the start- 
ing compound. The structure of the vitamin is given here: 

H H H 

3 H 
HC— C-C-C: 
H 





II 

-C-C 



H 



0- 



Vitamin C (Ascorbic Acid) 

Ascorbic acid is easily oxidized and reduced by virtue of the di-enol group. Mild 
oxidation or mild reduction does not affect the physiological properties to any 
marked extent. Severely oxidative procedures or irreversible oxidation and reduc- 
tion will destroy the ant i- scorbutic potency. 

Good sources of vitamin C are young sprouting plants, and it is not surpris- 
ing therefore that a study of these materials should furnish some information on 
the origin and physiology of vitamin C. Animal experimentation has of necessity 
been limited to the study of man, monkeys, and guinea pigs, since it has not been 
shown that other species require this particular vitamin. It is of interest in 
this connection that a study of chickens and rats ~bj Bessey and King (2) demon- 
strated that the vitamin C level in the tissues of these animals did not vary 
from that found in the guinea pig and other animals which are not able to synthe- 
size the vitamin. It is thus apparent that both chicks and rats are able to syn- 
thesize vitamin C. 

Vitamin C is in some way related to the actions of certain enzymes in the 
tissues, but no specific relationships have been demonstrated. Indirect evidence 
lends credence to the possibility that the action of the vitamin is in a large 
measure due to the oxidation-reduction powers of the compound (6,26). The theory 
that vitamin C serves primarily for the purpose of transporting hydrogen or acting 
as- a respiration catalyst has been questioned by Stotz, Harrer, Schultze and King 
(38). There have been reports that ascorbic acid has both activating and inhibit- 
ing effects on enzymes in vitro. These have been summarized by King (29). Ascor- 
bic acid has been shown by Green and Richter (19) to act as an inhibitor in the 
adrenalin-adrenochrom oxidation system in heart muscle. Lemberg and associates 
(31) have reported on a coupled oxidation between ascorbic acid and hemochromo- 
gens. Ecker et al (13) have found a relationship between vitamin C and guinea 
pig blood complement. This finding appears to be important since it furnishes 
some light on the oxidation reduction behavior of complement. 



43- 



kk 

Pathology of Vitamin C 

A vitamin C deficiency exemplifies itself in a number of ways, as shown "by 
an anemia, a decrease in resistance, injury due to toxins, etc. More detailed 
examination of the deficiencies indicates that there are morphologic changes in 
the capillaries, in the connective tissue and in bone. Gross changes can he seen 
in the various parts of the skeleton, especially in the costochondral .junctions. 
The teeth are often affected in people having scurvy, since the dentine is "pitted 1 ', 
and the cement is weakened. The tooth changes are also seen in the incisor teeth 
of the guinea pig and monkey. Of the other mouth changes, perhaps the most common 
is the lesion of the gingiva. Disintegration of the mucous membrane with concur- 
rent ulceration and infections arc the symptoms one usually finds in the gums. In 
suvere cases of scurvy there is edema with blood accumulations in the serous 
cavities due to capillary fragility. This manifests itself also by petechial 
hemorrhages in the skin. The eyes and muscles also show bloody infiltrations, 
and there is general circulatory collapse (11). 

Vitamin C Thera peutics 

The vitamin C reserves of the new born infant are a direct reflection on the 
state of nutrition of the mother (7,8). The rapid decrease in the ascorbic acid 
content of the blood of the new born infants within the first 10 days is an indi- 
cation that the vitamin C reserves supplied at birth must be augmented either by 
breast milk or by a supplement to artificial feeding. The ascorbic acid require- 
ment of very young infants ha3 been set at 5 "to 15 milligrams per day according to 
the recommendation of the Technical Committee on Nutrition of the Health Organiza- 
tion of the League of Nations (36). Although many investigators have studied the 
saturation and excretion habits of various age groups, little is known about the 
ascorbic acid requirement of children. The requirements of adults for this vita- 
min are not definitely known, but in general the results indicate that between 60 
and 100 milligrams of the vitamin are necessary for a 60 kilogram adult (23,^-2). 
Slightly higher amounts have been shown to be required by pregnant and lactating 
women (35)- 

The tentative dietary requirements recommended by the Foods and Nutrition 
Committee of the National Research Council for vitamin C have been arrived at by 
more than fifty authorities who were consulted on this subject. The figures are 
for an average individual in a particular age group. The adult man or woman re- 
quires 70 to 75 milligrams of vitamin C. A pregnant woman's allowance was set at 
100 milligrams, while a lactating mother's requirement was 150 milligrams per day. 
The infant under on? year needs 30 milligrams a day according to the suggestions 
of this Committee. Preschool children from 1 to 5 years should get 70 milligrams 
a day. Adolescent girls need approximately 75 while the adolescent boy needs 
nearly 100. The age of the individual is a consideration in estimating the re- 
quirement as well as the expected activity of the individual. 

Methods of Assay for Vitami n C 

The development of suitable diets for the assay of vitamin C were in a large 
measure due to the work of Hoist and Frohlich (25), Cohen and Mendel (10 ) and 
La Mer, Campbell and Sherman (JO). These workers used the guinea pig as the ex- 
perimental animal and measured antiscorbutic activity. Harris, Mills and Innes 
(21) have described a curative technic also using the guinea pig. This method has 
the advantage of economy in experimented animals since the guinea pigs can be 
depleted and fed another supplement. A tooth method first proposed by Hojer (2^) 
depends upon the degree of scurvy that can be estimated by histologic examination 



^5 

of the teeth. This worker described ten different stages in the scorbutic tooth, 
and by the recognition of the disorganization of the odontoblasts and dentine 
structure, a reliable index of scurvy could be obtained. It has the disadvantage 
of any histologic method in that much time and effort is necessary to clearly 
differentiate the various stages of the pathology. 

The chemical methods are to be desired over biologic methods for the obvious 
reason of time economy. The original methods proposed by Tillmans, Hirsch and 
Hirsch (kl), Harris and Ray (22) and Bessey and King (2) utilized the titration 
of the vitamin with 2,6 dichlorophenolindo phenol. Various modifications have been 
proposed for its use in plant materials and in animal tissues (2). The develop- 
ment of the photoelectric colorimeter has now increased the accuracy of the titri- 
metric procedure, and by carefully controlling the time of the reaction, repro- 
ducible figures are obtained (lU,33)» Other chemical methods are based on the 
reduction of ferricyanide, phosphotungstic acid, and phosphomolybdic acid as well 
as other color reagents. The results obtained with these reagents are not as 
fully satisfactory as those obtained by the dichlorophenolindophenol titration,- 
but the reason for this is not clear. It appears from most of the evidence that 
the dye method is superior for determining vitamin C in blood, urine, plant and 
animal tissues (l). 

Vitamin C in Animal Tissues 

The vitamin C content of many foods has been summarized in several recent 
publications (U,5,12) but very little has been tabulated with reference to the 
vitamin C content of meats and meat products. In Table TV the available values 
on the vitamin C content of meats are given. It is apparent that of the animal 
tissues adrenals are by far the best source of vitamin C, and it is clear why the 
biochemists chose the adrenals as their source material for the isolation of the 
vitamin. In general, the ascorbic acid content of the adrenals from most domestic 
animals lies in the same range, from 100 for the calf adrenal to 15 milligrams 
per 100 grams of the tissue for rabbit and chicken. The adrenals of the pig, ox, 
sheep, cow and steer show a value within this range. McIIenry and Graham (32) 
found that after cooking, their sample of cow adrenals had an increased ascorbic 
acid content. This observation is borne out by the data of Rudra (37) ""ho also 
found that the sample of ox adrenal contained l'+l milligrams per 100 grams of 
ascorbic acid whereas the uncooked adrenal contained 125 milligrams per cent. 
This discrepancy is explained in part by the failure to take into account the dry 
basis of the material that was analyzed. The cooked adrenals had evidently lost 
moisture during the cooking process and after removal from the utensil further loss 
of moisture occurred. 

The thymus is the tissue which shows the second highest vitamin C content 
although it is appreciably lower than that of the adrenals. Glick and Biskind 
(16,17,18) found a range of values for cow and steer thymus. The cow thymus 
samples ranged from 35 - 52 mg. per cent, while the steer samples of the gland 
varied from k.O to I7.0 mg. Fujita and HJbihara (15) also found beef thymus to 
be fairly high in comparison to other tissues which they assayed, the sample yield- 
ing k2 mg. per cent. 



A number of workers have reported the vitamin C content of the liver of 
various species. Bessey and King (2) gave 28.0 mg. per cent for chicken liver 
while Munsell (3^) gave I7.5 mg. per cent. Chi and Read (9) reported 13.0 mg. for 
a sample of duck liver which they analyzed. Guha and Ghosh (20) found 5-3 WE- per 
cent in pigeon liver. Bessey and King (2) claimed that rabbit liver had 'I3.0 mg. 
per cent which compared favorably with the rabbit liver analyzed by Tauber and 
Kleiner (kC), but did not agree with the work of Birch and Dann (3) which gave ^0 
mg. per cent. Munsell (3*+) gave 37.5 mg. for beef liver, 32.0 mg. for calf liver, 



he 

and 37-5 W>- for lamb liver, and 26.0 mg. for pig liver. Bessey and King (2) 
found 19«0 mg. for hog liver and Birch and Dann (3) found 38. mg. for 100 grama 
of this tissue. These last workers claimed that sheep liver "was a fairly high 
source since they found 1+5-0 mg. per cent, and this high content is verified "by 
Svirbely (39) who found kS.O mg. for 100 grams of this same species of liver. 
Svirbely found that this sample of pig liver contained 12.0 mg. per cent while ox 
liver and calf liver contained 31 an & 33 respectively. Rudra (37) found goat's 
liver to he materially affected in its vitamin C content of the sample of goat's 
liver was 73 -^ ^6- P er cent, while after cooking it was found to contain 27-3 ^g* 
If the same sample were then subject to "easy cooking" much less was lost, the 
value being 55 • 5 ^6- P er cent. When the same sample was fried rapidly it contained 
53.2 mg. per cent. This is difficult to explain since Rudra reported that the 
sample of ox adrenal which he cooked gave an apparent increase in its vitamin C 
content. Unlike the adrenal, the goat's liver was adversely affected in its 
vitamin C content after processing. 

Chicken kidney contained 15.0 mg. according to Bessey and King (2). These 
workers found 7.0 mg. for rabbit kidney, which is exactly that found by Tauber and 
Kleiner (ho) for their sample of rabbit kidney, but Birch and Dann (3) found 25-0 
mg. per 100 grams of this tissue. Chi and Read (9) found 14.0 mg. per cent for 
hog kidney. 

The sample of chicken heart analyzed by Bessey and King (2) contained 3.0 mg. 
per cent while the sample of hog heart analyzed by these workers gave ^.0 mg. per 
cent, and the rabbit hea.rt showed 5»0 mg. per cent. Beef heart contained 0.1 mg. 
per cent according to the figures given by Fujita and Ebihara (15). Chicken muscle 
also gave a low value of ^-.0, which wa3 identical with that of rabbit muscle. No 
values on the vitamin C content of beef muscle are given in the literature, but 
certain physiological studies (h^,kk) have presented data which indicate that 
muscular tissue contains small amounts of the vitamin. Human tissues [k-6) have 
furnished some indication that both the skeletal and the striated smooth muscle 
have small amounts of vitamin C . 

Chicken brain contains a fair amount of vitamin C as shown by the work of 
Bessey and King (2) and by Fujita and Fbihara (.15). These workers have given 
values of 33-0 milligrams for chicken brain, 22.0 mg. for rabbit brain and 18.0 
for hog brain. The Japanese workers have given the value 12.0 milligrams for 
the white matter and 16.2 for the gray matter of beef brain. Fujita and Ebihara 
(15) gave 5.7 for beef spleen, and Tauber and Kleiner (ko) gave 15.0 for rabbit 
testes and 30.0 milligrams per cent for rabbit spleen. Tauber and Kleiner (^-0) 
also gave 30 milligrams per cent for rabbit pancreas while the Japanese workers 
reported 10.6 for beef pancreas. 

Much of the experimental work dealing with the physiology, pharmacology, and 
pathology of vitamin C action has furnished additional values on the vitamin C 
content of animal tissues. Only that literature which pertains directly to pos- 
sible human consumption at present is included in the table. There are a variety 
of reports which deal with the vitamin C content of certain portions of a particu- 
lar gland, especially the adrenal. Thu work of Glick and Biskind (16, 17,18) is 
largely limited to this consideration, and these workers found that the vitamin 
C was localized in the fascicular region of the adrenal gland. 



There appears to be considerable variation in the results reported by the 
various workers, but here sg;ain, the influence of the ration, conditions of the 
experiment, and the method should be consulted before undue criticism is given to 
a particular value. Liver is a good source of vitamin C in animal tissues and com- 
pares favorably with the amounts found in the thymus. Liver contains less vitamin 



C than the adrenals. There is a surprisingly good agreement with respect to the 
ascorbic acid content of the adrenals from the different species. The adrenals 
are about J ix> K tines as rich in vitamin C as the livers, "but the muscular tis- 
sues from the several species are only one-tenth as rich as the livers. 

Meats must then be considered as only a fair source of vitamin C, and 
although there appears to be little destruction during cooking as shown by a few 
workers, our other foods are better able to supply this factor. 



TABLE IV 
Vitamin C Content of Animal Tissues 



Meat 



Milligrams ascorbic 




acid per 100 grams 




fresh weight 


Eeference 


31.0 


39 


37-5 


3^ 


32.0 


3^ 


33.0 


39 


19.0 


2 


38.0 


3 


12.0 


39 


26.0 


3k 


37.5 


3^ 


1+5.0 


3 


ke.o 


39 


73.1+ 


37 


27.3 


37 


55.5 


37 


58.2 


37 


17.5 


3h 


28.0 


2 


13.0 


9 


5.3 


20 


13.0 


2 


1+0.0 


3 


10.0 


^0 


1^.0 


9 



Beef 


liver 


1! 


11 


Calf 


liver 


1! 


u 


Pig • 


Liver 


it 


it 



Lamb liver 

Sheep liver 
it it 

Goat liver 

Cooked goat liver 

Mild cooked goat liver 

Fried goat liver 

Chicken liver 
it n 

Duck liver 
Pigeon liver 
Rabbit liver 



Pig kidney 



48 



Meat 

Rabbit kidney 
ii it 

Chicken kidney- 
Beef adrenal cortex 
Cow adrenals 
Cooked cow adrenals 
Ox adrenal 
Calf adrenal 
Ox adrenal 

Ox adrenal after cooking 
Hog adrenal 
Pig " 
Sheep " 

Rabbit " 
it ii 

Chicken adrenal 
Cow thymus 
Steer thymus 
Beef thymus 
Beef heart 
Pig heart 
Chicken heart 
Rabbit 
Beef spleen 
Rabbit spleen 
Beef brain, white 

" , gray 
Pig brain 
Rabbit brain 
Chicken brain 
Beef pancreas 
Rabbit pancreas 
Lean beef muscle 
Chicken muscle 
Rabbit " 
Rabbit testes 



Milligrams ascorbic 






acid per 100 grams 






fresh weight 


Reference 




7.0 


2 




25.0 


3 




7-0 


40 




15.0 


2 




108.0 


15 




121.0 


32 




Hn.o 


32 




76.0 


39 




99.0 


39 




125.0 


37 




14.1.0 


37 




146.0 


2 




115.0 


39 




133.0 


39 




150.0 


40 




135.0 


2 




153.0 


2 




55-52 


16,17 




4.0-17.0 


16,17 




42.2 


15 




0.1 


15 




4.0 


2 




8.0 


2 




5.0 


2 




5.7 


15 




30.0 


4o 




12.0 


15 




16.2 


15 




18.0 


2 




22.0 


2 




33.0 


2 




10.6 


15 




30.0 


40 







3^ 




4.0 


2 




4.0 


2 




15.0 


40 





k9 

LITERATURE CITED 

1. Bessey, O.A. "Vitamin C, Methods of Assay and Dietary Sources". The Vita- 
mins, A Symposium, 1939- Am. Med. Assoc. : 

2. Bessey, O.A. and King, C.G. The Distribution of Vitamin C in Plant and 
Animal Tissues and Its Determination. ■ J. Biol. Chem. , 103, 687 (1933)- 

3. Birch, T..W. and Dann, W.J. Estimation ajid Distribution of Ascorbic Acid 
(Vitamin C) and Glutathione in Animal Tissues. Nature, 131, k-69 (1933)* 

k. Boas-Fixsen, M.A. .; The Vitamin Content of Human Foods as Affected by Process- 
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5. Boas-Fixsen, M.A. and Roscoe, M.H. Tables on the Vitamin Content of Human 
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5. Borsook, H., Davenport, H.W., Jeffreys, C.E., and Warner, R.C. The Oxidation 
of Ascorbic Acid and its Reduction in Vitro and in Vivo. J. Biol. Chem., 
Ill, 237 (1937). 

7. Braestrup, P.W. Studies of Latent Scurvy in Infants: III. The Content of 
Reduced Ascorbic Acid in Blood Plasma in Infants, Especially at Birth and in 
the First Days of Life. J. Nutr., 16, 363 (1938). 

8. Braestrup, P.W. Studies of Latent Scurvy in Infants: I.' Capillary Resist- 
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9. Chi, Y.F. and Read, B.E. Vitamin C Content of Chinese Foods and Drugs* 
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10. Cohen, B. and Mendel, L.B. Experimental Scurvy of the Guinea Pig in Rela- ■■ 
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11. Dalldorf, G. "The Pathology of Vitamin C Deficiency", The Vitamins, A Sym- 
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12. Daniel, E.P. and Munsell, H.E. "Vitamin Content of Foods". U. S. DeptAgr., 
Misc. Publication, 275, 1937- ''■ 

13. Ecker, S.E., Pillemer, L. , Wertheimer, D. and Gradis, H. Ascorbic Acid and 
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Ik. Evelyn, K.A., Malloy, H.T. and Rosen, C. Determination of Vitamin C (Ascorbic 
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50 

18. Glide, D. and Biskind, G.R. Studies in Histochemistry. IX. The Quantitative 
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19- Green, D. E. and Richter, D. Adrenaline and Adrenochrom. Biochem. J'., 51, 
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km 



51 



36. Report by Technical Commission on Nutrition on Work of the 3rd Session Bull. 
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of Man. Acta. brev. neerland, 6, 86 (1936). 

kj. Wachholder, K. , Anders, K. and Uhlenbrooch, K. Further Investigations on the 
Determination of Glutathione and Ascorbic Acid Content of Animal Tissues. 
Zeit. Physiol. Chem., 233, l8l (1935). 

kk. Wachholder, K. and Podesta, H.H. Variations in the Vitamin C Content of 

Different Muscles as Related to Their Oxidizing and Reducing Activity. Arch, 
ges. Physiol. (Pf lugers ), 258 , 615 (1937). 

lj-5- Wolbach, S.B. Pathological Changes Resulting from Vitamin Deficiency. J. Am. 
Med. Assoc, 108, 7 (1937). 

k6. Yavorsky, M., Almaden, P. and King, C.G. Vitamin C Content of Human Tissues. 
J. Biol. Chem., 106, 525 (193*0 • 



The discussion up to this point has concerned itself with 
those vitamins which are not members of the vitamin B group. 
The material to follow is a survey of the data obtained in 
this laboratory on the distribution of the various members of 
the B complex in animal tissues during the past several years. 
The figures which will be given are also appraised in the light 
of the investigations of other workers. By way of introduction, 
the preparation of the samples which were used in our work is 
described together with the proximate analyses of these tis- 
sues. A separate chapter is devoted to each of the vitamins. 



■53- 



CHAFTER VII 
PREPARATION OF THE SAMPLES 



CHAPTER VII 
PREPARATION OF THE SAMPLES 

The assay of a large number of animal tissues involves considerations of 
storage, the age of the meat when the housekeeper "buys it, the effect of the age 
of the animal on the vitamin content, and the effect of cooking on the nutritional 
value. In most cases the tissue used for analysis was obtained from the local 
packer "but some samples were obtained from a local meat market. The prime or 
choice grade of beef, veal, lamb or pork was usually used for the assay. In 
several attempts to determine whether the grade of the carcass was a factor in- 
fluencing the vitamin content, we obtained several grades, taken from animals 
judged as poor, medium and excellent. Some of the samples of kidney, liver and 
muscle were kept in the frozen state for certain periods before being reassayed 
to determine any loss due to storage. The dry samples were also stored before 
making another assay on the same sample. 

In the early part of the investigation an attempt wa3 made to obtain infor- 
mation on the nutritional history of the animal so that it might be possible to 
correlate the vitamin content with the type of feed consumed by the animal. How- 
ever, this line of endeavor was relinquished since a limited amount of such in- 
formation could be ascertained. The animals brought to the packer came from many 
districts which made it impossible to obtain any accurate information on the 
quality of the feed and feeding habits. The majority of the samples used were 
prime or choice cuts from the various species. 

The great number of samples that were to be assayed made it necessary to 
consider drying the fresh tissue so that it might be preserved with greater ease 
and with insurance against spoilage. Many of the samples were to be assayed for 
several of the vitamins thus requiring that the sample be kept for an indefinite 
period. In order to develop a method for the preservation of the samples, it 
was essential that a procedure be used that would give a minimum loss of the vita- 
mins. The available information on the stability of these accessory food factors 
indicated that thiamin was the least stable vitamin. It appeared logical to us 
that any method directed towards preserving this substance would in all probabil- 
ity retain all the other vitamins. It was very necessary therefore to determine 
the possible destruction of this vitamin under different temperatures in the 
presence and in the absence of air. Such preliminary work indicated that tissues 
which were dried even in vacuum at temperatures above 70°C showed some destruc- 
tion of thiamin. In order to determine if any destruction of vitamin B-, occurred 
during careful drying at room temperature or slightly above, samples of pork from 
the same animal were assayed in the fresh and in the dried condition. For this 
particular test, both the loins and the hams were used. The fresh samples to be 
fed daily were kept in covered containers and stored in a freezing unit of the 
refrigerator. The meat which was to be fed in the fresh condition was kept frozen 
in the refrigerator until needed and only small amounts were kept for a few days ' 
feeding. Care was taken to reduce the time required for weighing out the daily 
samples that were to be fed to the rats . No difficulties were encountered in 
feeding the samples since the fresh meats were consumed immediately by the rats. 

A portion of each sample of meat was dried and the fat, connective tissue, 
and bone were removed as far as was possible. The trimmed muscle was cut into 
small pieces and put through a meat grinder. At this point enough of the wet tis- 
sue was taken for a moisture analysis, usually 25-35 grams. 



•55- 



55 

After grinding, the meat was spread out on galvanized pans, 30 x 30 inches, 
in layers about three to four millimeters deep. Some tissues did not lend them- 
selves to easy spreading, and it "was necessary to repeat the grinding in order to 
obtain greater uniformity. In all cases, the meat was spread as thin as possible 
in order to facilitate the drying and thus minimize the destruction of the vita- 
mins. The juice which seeped away from some of the ground tissue was dried down 
with it. A thermometer was placed in each pan and all of a particular tissue was 
dried at the same time in the drying chamber. A continuous current of warm air 
was circulated over the pans, and by this means the meat was dried at a tempera- 
ture range of 35 "to 50°C. Rarely did the temperature rise above this. Usually 
the time required for drying was between six and ten hours, but at times it was 
necessary to keep the sample in the pans for as long as 20 hours if the substance 
had a very high fat content. It was necessary to turn the samples at least once 
to shorten the period of drying. After drying, the samples were ground in a 
plate grinder, with the least shearing strain possible in order to minimize the 
heat produced. Many of the samples were able to pass a 50 mesh screen, but cer- 
tain tissues having a high connective tissue content were further ground in an 
automatic mortar so that the sample was able to pass the 80-120 mesh screen. 
Samples high in fat were carefully treated so as to obtain the finest state of 
subdivision without affecting the fat content. 

To determine if any vitamin B. was destroyed during this drying process both 
the fresh and the dried meat were assayed simultaneously. M^re complete results 
on this parallel experiment will be given under vitamin B-, assays, but it will 
suffice here to say that very little if any of the vitamin was destroyed by this 
method of drying. Similar studies on drying procedures were made at the time of 
the vitamin B< assays . Here again fresh meat was fed with the basal ration and 
compared to the same sample fed in the dry state. After a number of experiments 
it was concluded that there was no destruction of vitamin B^ in our method of 
drying the tissues. It should -be pointed out that the majority of the samples 
were always kept in brown bottles and in diffuse light or darkness to minimize 
the possible destruction of riboflavin. 

The method of drying described above was then followed throughout the pro- 
ject since it was apparent that careful treatment of the meat would not affect 
the vitamin content. Although many of the samples were assayed for several of 
the vitamins, there were a number of samples which were obtained for a single 
experiment and were thus used completely. At other times, some samples were used 
at the beginning of an experiment, but were replaced during the period by another 
sample of the same tissue either because the first sample wa3 exhausted or for 
some other reason. 

A portion of our experiments were designed to obtain some information on 
the effect of household cooking procedures on the vitamin content of meat. The 
samples which were used in the bioassay of household cooked meats were ordinary 
cuts of meat such as are sold on the open market. Usually, the attempt was made 
to prepare the sample in such a manner that part of it could be dried immediately 
and assayed for its vitamin content before cooking, and the rest of the same tis- 
sue could be either roasted, fried, broiled, or stewed. In some cases a part 
of the same carcass was subjected to all of these procedures. Each of the samples 
was prepared and cooked under ordinary household conditions. The time of cooking, 
the temperature of the process, the size of the cut, the weight of the cut, and 
other necessary information was gathered before arid after each portion was cooked. 
The information on temperature is necessarily limited in some procedures since no 
accurate data could be obtained on the internal temperature of fried meats. The 
types of thermometers available for this purpose furnish at best only a crude 
approx imat ion . 



57 

The cooked samples which were used in the different assays are described in 
the following paragraphs. The sample numbers are given so as to facilitate the 
discussion in succeeding chapters since several of the cooked meats were assayed 
for several of the vitamins. 

Sample 37 Baked Pork Chops 
Sample 38 Fried Pork Chops 
Sample 39 Roast Loin of Pork 

These samples were obtained from the same loin of a hog. Eighteen average 
sized pork chops were cut out from the center of the loin; one-half of these were 
fried, the rest were baked. The tenderloin end was cut off and roasted. The re- 
maining part of the loin was trimmed and kept as a control on the cooking proce- 
dures. The chops were fried in an open pan on top of the stove for 10 minutes 
until they were medium done. The meat that was roasted was first seared in the 
oven for 15 minutes at 290°C . , then 1 and l/2 cups of water were added and the 
roast was covered and allowed to cook for 80 minutes at l85°C . The chops which 
were baked were seared for 10 minutes on top of the stove. One and one-half cups 
of water were then added and the meat kept at a temperature of I85 for 55 minutes, 
being covered during the baking. 

Sample kO Untreated Beef Round 

Sample kl Broiled Round Steak 

Sample k2 Fried Round Steak 

Sample 1+3 Roasted Round of Beef 

The broiled round steak was a portion of the sirloin tip and eye of round. 
It was placed in the oven for 20 minutes at a temperature of 550°F. (29^°C ). The 
fried top round steak was fried over a medium flame for 20 minutes. The sample 
of bottom round was roasted by searing for 20 minutes in an uncovered roaster at 
55° F., the meat was then covered and roasted at 35°°F (117°C ) for 2 hours. 



Sample kh Uncooked Veal Hindquarter 
Sample U5 Fried Veal Chops 
Sample kS Roasted Leg of Veal 

The fried veal chops were prepared by frying over a medium flame for 20 
minutes in a covered pan. The roasted veal was seared for 20 minutes at 55°°F. 
(288°C. ) and cooked in a covered roaster for a total of two hours at 177°C ■ 



Sample k'J Uncooked Smoked Ham 
Sample 48 Fried Smoked Ham 

Slices of approximately 3/3 inch were fried for 15 minutes in an open frying 
pan over a medium flame. 



Sample 4 9 Uncooked Beef Kidney 
Sample 5° Stewed Beef Kidney 



The neat was freed of all adhering fat and cut into one inch cubes. The 
cubed meat was placed in a saucepan with water and allowed to simmer for 4 5 
minutes. The same volume of water was maintained throughout the cooking process 
The cubes were removed from the liquid and dried as usual. 



58 

Sample 51 Untreated Fresh Ham 
Sample ^2 Fried Fresh Ham 

The fresh ham was cut into half inch slices in the usual manner and fried 
in an open pan for approximately 15 minutes. 



Sample 53 Uncooked Beef Heart 
Sample 5U . Stewed Beef Heart 

This sample of cooked meat was prepared in exactly the 3ame manner as the 
sample of stewed beef kidney. 



Sample 84 Uncooked Beef Kidney 
Sample 85 Stewed Beef Kidney 

These were prepared in a similar manner to the previous kidney sample. 



Sample 92 Uncooked Pork Loin 
Sample 93 Fried Pork Chops 
Sample 9^ Roasted Jxiin of Pork 

Another pork loin was similarly treated except that in this trial the baking 
of the chops was left out. The entire loin weighed 12.5 pounds and was so cut 
that a piece weighing about k pounds was cut off the tenderloin end for roasting, 
and another piece weighing about 2.5 pounds wa3 cut into medium sized chops for 
frying. The rest of the loin was trimmed and dried. The roast was seared for 15 
minutes in an uncovered roaster at 55°°C., then 2 and l/2 cups of water added, the 
roast covered and heated for one hour and forty-five minutes at 375 oF » The fried 
loin chops were placed in a covered pan and heated over a medium flame for 15 to 
20 minutes. 



Sample 95 Untreated Pork Ham 
Sample 101 Boiled Ham 
Sample 102 Smoked Ham 

The boiled ham was cured by the local packer in the regular manner, then 
cut up and made into a loaf which was placed in water at a temperature of about 
160 F. and kept there until the temperature within the meat reached 1^2°F. (6l°C). 
The smoked ham was a picnic ham which was cured and smoked by the same local 
packer. 



Sample 98 Untreated Beef Liver 
Sample 99 Fried Beef Liver 

The liver was cut into l/k to 3/8 inch slices and fried for 20 minutes over 
a slow flame. 



59 



Sample 105 Uncooked Beef Round 
Sample 105 Fried Beef Round 
Sample 107 Roast Round of Beef 

These samples were prepared In a manner similar to samples kO to hh. 



Sample 108 Uncooked Beef Spleen 
Sample 3 09 Stewed Beef Spleen 

Another sample treated like kidney or heart. 



Sample 110 
Sample 111 
Sample 120 
SaniDle 121 



Uncooked Beef Liver 
Fried Beef Liver 
Uncooked Beef Liver 
Fried Beef Liver 



Similar to 98 and 99. 



Sample 1^-0 Uncooked Pork Ham 
Sample lUl Fried Pork Ham 
Sample 1^2 Roast Pork Han 

This sample of ham was cut into half inch slices and fried until "medium 
well done" over a medium flame. The time of frying was approximately 20 minutes 
The shank portion of the ham was roasted in a medium oven for two hours without 
searing. 



Sample 1^+5 Uncooked Beef Round 
Sample lkf Broiled Beef Round Steak 
Sample llj-8 Fried Beef Round Steak 

The steak slices were about 3/8 to 1/2 inch in thickness and were broiled 
beneath an electric grill for 15 minutes with occasional turning of the meat. 
Each side was turned towards the flame twice. The slices that were fried were 
the same thickness as the broiled steaks. The frying was done over a low flame 
for 15 minutes and turned twice . The small amount of fat added to aid the cook- 
ing procedure was sponged off the fried steaks. 

The cooked samples were dried at the same time and under the same conditions 
as were the untreated tissues . 



CHAPTER VIII 



PROXIMATE ANALYSIS OF THE ANIMAL TISSUES 



Proximate Analysis of Meat Samples Under 



Investigation 



Literature Cited 



Page 

62 

.69 






BBHBI 



CHAPTER VIII 

PROXIMATE ANALYSIS OF THE ANIMAL TISSUES 

Muscular tissue together with epithelia, connective tissue, nervous tissue, 
and. the fluid tissues, blood and lymph, make up the five fundamental tissues of 
the "body. The ordinary muscular fibers consist of proteins, inorganic salts, 
water and unknown organic materials which play a part in the normal function of 
the muscle fibril. The majority of muscle protein is an albumin known as myosino- 
gen or myosin. Muscle hemoglobin also exists to a small extent. Connective tis- 
sue occurs in muscle and acts as part of the framework which holds the individual 
muscle fibers together. This connective tissue is collagen, a protein which 
yields gelatin on partial hydrolysis. Fat also occurs between the individual 
fibers. Carbohydrate occurs to a small extent in the muscle. 

Analyses of animal tissues were done as early as 1859 by Laves and Gilbert 
(7) and by Jordan (5). A great number of such analyses were done by Haecker {k) } 
Moulton, Trowbridge and Haigh (8), Tschirwinsky (11 ), Soxhlet (9), Weiske and 
Wildt (13), Wilson (1*0, Washburn and Jone3 (12) among many others. More recent 
work by Toscani, Rupp and McClellan (10) gave values for the individual muscle 
meats and organ tissue. 

The analyses of Lawes and Gilbert (7) for the entire carcass of domesticated 
animals showed that for a moderately fat beef, the muscle was ^8 per cent, bones 
11. h per cent, fat 12.5 per cent, and the skin, etc., 31*1 P er cent. These can 
be taken as representative figures for the proportion of muscle, bone, fat, skin 
and entrails. Some early work on the analysis of the entire animal carcass was 
done by Konig (6) and by Moulton, Trowbridge and Haigh (8). Typical analyses are 
given in Table V to illustrate the variation one might expect in such analyses. 



TA3IE 7 
Typical 7ariation in Carcass Analysis 







Protein 


Ether Ext. 






Animal 


N x 6.25 


Fat d P 


Water $ 


Konig (6) 


Ox 


21.0 


5^ 


72.0 




Cow 


19.1 


7.7 


71. c 




Calf 


19.0 


7.* 


72.0 




Sheep 


15.6 


28.6 


53.0 




Pork 


1^.5 


37-3 


1+8.0 


Moulton, 


Beef 


19.0 


Ik.k 


62.0 


Trowbridge, 










and Haigh (8) 


Sheep 


19.9 


13. If 


61.0 




Pork 


11 A 


32.0 


56.0 



There is some discrepancy in the two sets of figures, especially in the fat 
content. The water analysis is thus affected and the only comparable figures are 
the protein values. It should be remembered too, that the animals analyzed were 
quite different in feeding habits, care and so on, and this would all be reflected 



-51- 



_J 



62 

in the state of nutrition of the animal and therefore in the analyses. A summary 
of the composition of meat has been prepared by Winton and Winton (15 ) who cite 
the work of many investigators. Atwater and Bryant are quoted extensively since 
the first work in this country at the Connecticut Experiment Station dealt with 
this problem. 

The proximate analyses of the animal tissues which were used throughout our 
study were recorded soon after the tissue was prepared for assay purposes. Nearly 
150 animal tissues were analyzed for their moisture content, crude fat, crude pro- 
tein by the methods described below. Table VI lists the complete proximate analy- 
ses on all the tissues which were used in the assays. 

Proximate Analysis of Meat Samples under Investigation 

The moisture determinations were made on the sample of the fresh tissue taken 
just before drying. These determinations were made according to the method of 
the Association of Official Agricultural Chemists (2). The wet tissue was weighed 
out on a piece of filter paper and placed inside the flask of the apparatus of 
Bidwell and Sterling. The toluene was added so that it covered the material in 
the flask. The distillation was carried out to completion as indicated by the 
clear column of water in the graduated arm. A glance at the determinations shows 
that the moisture content of most of the animal tissues averages about 75 P er cent. 
In all cases there was less moisture in the treated meat3 such as 3moked ham, 
tenderized ham, and boiled ham. The meats which were cooked al30 showed a los3 
in moisture as would be expected since the water was lost in the cooking process. 
Those samples such as beef pancreas and beef brain, which had a high fat content 
also showed a lower moisture content. The livers as a rule had less moisture than 
did the muscle tissue. 

The protein determinations were made by determining nitrogen by the official 
method using the Kjeldahl -Gunning -Arnold method (3). The value for nitrogen was 
multiplied by 6.25 thus giving the approximate protein content of the dry meat 
sample. It was thought best to determine nitrogen on the dry sample so that more 
uniform results could be obtained. The protein content of the samples could thus 
be compared since the influence of moisture variation was at a minimum. The pro- 
tein content varied between U2.5 per cent for a sample of brain and Qk.k per cent 
for a sample of chicken muscle. The majority of the meats contained between 65 to 
75 per cent of crude protein on the dry basis. The variation in protein content 
can be attributed to the different nutrition of the animals and to the varying 
fat content. 

The fat content of our samples was determined by making a crude ether extract 
on the dried sample. The method followed was that given in Official Methods of 
the Association of Official Agricultural Chemists (l). As would be expected, 
tissues such as beef pancreas, beef brain and beef tongue showed very high fat 
content. 

The analyses of the 150 or more meat samples are able to furnish information 
on several tissues which have previously had little recognition. The protein and 
fat percentages of the various tissues are expressed in terms of the moisture free 
samples. The presence of much fat in the tissue would of necessity lower the pro- 
tein, and yet have no appreciable influence on the moisture content. This is true 
for beef brain sample 13 since the fat percentage increases as the protein content 
decreases. Samples of muscle may also show this relationship. 



■■ 



-L 



63 



TABLE VI 
Proximate Analysis of Animal Tissues Used in These Studies 



Meat 


Process 


Meat 
sample 


Per cent 
Moisture 


Per cent Fat 
(ether ext. ) 

in dry 

tissue 


°jo Protein 
(Nx5.25) 
in dry 
tissue 


Lamb muscle I 


Tried 


1 


— 


28.0 


8).k 


Lamb muscle II 


:i 


2 


-- 


29.3 


63-9 


Lamb muscle III 


M 


3 


-- 


23.2 


69- k 


Beef spleen 


ii 


k 


73-6 


17.0 


68.5 


Calves liver 


It 


5 


76.7 


5-0 


71.5 


Beef lung 


it 


5 


77-5 


8.7 


76.9 


Beef sweetbreads 


if 


7 


72.2 


37.0 


1+6.7 


Lamb kidneys 


|! 


8 


69-9 


13.6 


59.^ 


Beef kidney 


(I 


9 


7^.0 


28.7 


59.9 


Beef liver 


II 


10 


72.0 


Q.k 


66.5 


Beef brain 


II 


n 


8o.o 


1)2.7 


k$.9 


Pork muscle 


It 


12 


7^-5 


13.8 


78.2 


Beef heart 


II 


13 


73-0 


2^.9 


56.0 


Veal hindquarter 


II 


ik 


75-0 


Ik. 9 


76.1+ 


Veal hindquarter 


M 


15 


73-0 


17.5 


70.7 


Veal hindquarter 


it 


15 


55-0 


22.2 


55-5 


Beef muscle III 


H 


17 


55-0 


32.2 


59-2 


Beef muscle II 


it 


13 


79-0 


28.55 


51.75 


Beef muscle I 


ii 


±9 


52.0 


1+3.0 


1+9.7 


Lamb muscle 


it 


20 


7^.7 


15.2 


75.78 


Lamb muscle 


i: 


21 


74.0 


17.2 


76.5 


Lamb kidney 


n 


23 


77.9 


18A 


69. ] + 


Lamb liver 


It 


2k 


69.7 


15.I 


56.3 


Pork kidney 


It 


28 


75.0 


19.8 


55.75 


Pork liver 


II 


29 


7J+.0 


10.6 


65.6 


Pork loin 


It 


30 


-- 


51.6 


kk.J 


Calves kidney 


It 


31 


78.0 


16. 7 


58.5 


Pork ham 


it 


32 


72.5 


19.O 


76.2 


Pork liver 


It 


33 


70.5 


13.8 


55.8 


Pork loin 


it 


3^ 


71.5 


2k. 3 


57. k 


Lamb leg 


II 


35 


78.3 


22.2 


72.6 


Pork loin 


it 


35 


68.8 


31.0 


5I+.5 



64 



TAB IE VI (continued) 





Meat 


Process 


Meat 
sample 


Per cent 
moisture 


Per cent Fat 
(ether ext. ) 

in dry 

tissue 


c /o Protein 
(Nx6.25) 
in dry 
tissue 


Pork 


loin 


Baked 


37 


78.5 


34.4 


59.3 


Pork 


loin 


Fried 


38 


76.0 


28.4 


62.8 


Pork 


loin 


Roasted 


39 


74.7 


30.8 


61. 3 


Beef 


round 


Dried 


40 


68.6 


16.6 


75-6 


Beef 


round 


Broiled 


hi 


52.0 


23.1 


73.0 


Beef 


round 


Fried 


42 


73-5 


16.6 


77-0 


Beef 


round 


Roasted 


h3 


62.0 


15.2 


78.6 


Veal 


hindquarter 


Dried 


kh 


71.0 


15.8 


79-2 


Veal 


hindquarter 


Fried 


k$ 


22.8 


14.8 


77-5 


Veal 


hindquarter 


Roasted 


45 


40.0 


4.2 


83.0 


Smoked ham 


Dried 


hi 


67-5 


14.4 


63.1 


Smokt 


;d ham 


Fried 


48 


52.5 


16.0 


52.7 


Beef 


kidney 


Dried 


49 


78.9 


13.1 


75.3 


Beef 


kidney 


Steve d 


50 


__ 


13.2 


72.4 


Pork 


ham 


Dried 


51 


7^.5 


21.2 


74.2 


Pork 


ham 


Fried 


52 


-- 


20.4 


76.5 


Beef 


heart 


Dried 


53 


77-5 


12.6 


78.2 


Beef 


heart 


Stewed 


5 4 


79.5 


11.3 


81.4 


Lamb 


leg 


Dried 


55 


72.3 


21.3 


74.1 


Veal 


hindquarter 


Dried 


56 


76.4 


12.4 


81.5 


Beef 


brain 


M 


57 


79.1 


48.6 


46.7 


Beef 


liver 


11 


53 


67.6 


18.3 


64.2 


Beef 


spleen 


11 


59 


76.8 


12.8 


77.4 


Lamb 


liver 


11 


61 


65-5 


23.5 


57-3 


Pork 


kidney 


V 


62 


73.4 


18.6 


72.3 


Beef 


lung 


It 


63 


77.9 


10.2 


75.9 


Beef 


pancreas 


M 


64 


67.6 


41.2 


56.4 


Light chicken 


It 


65 


72.8 


11.6 


84.4 


Lark 


chicken 


tl 


66 


74.2 


18.8 


75.2 


Beef 


tongue 


It 


61 


6S.k 


41.7 


54.5 


Light chicken 


M 


68 


75^ 


5.^ 


83.9 


Dark 


chicken 


It 


69 


76.4 


12.2 


8o.4 


Veal 


liver 


(1 


70 


73.5 


20.2 


65-2 



TABLE VI (continued) 



65 











Per cent Fat 


Jo Protein 










(ether ext. ) 


(Nx6.25) 






Meat 


Per cent 


in dry 


in dry 


Meat 


Process 


sample 


moisture 


tissue 


tissue 


Pork heart 


Dried 


71 


79-5 


lk.k 


70. k 


Tenderized ham 


«• 


72 


68.7 


10.8 


67-6 


Beef heart 


x 


73 


78.7 


Q.k 


73.6 


Pork loin 


<i 


7^ 


71.8 


2k.k 


66.2 


Veal hindquarter 


n 


75 


77-0 


-- 


81.5 


Beef spleen 


t! 


76 


-- 


9.0 


66.3 


Beef "brain 


tt 


77 


-- 


lf5-3 


kk.e 


Beef lung 


ft 


78 


— 


10.6 


13-h 


Beef pancreas 


II 


79 


-- 


19.2 


66.6 


Lamb leg 


II 


80 


7^.2 


1J+.0 


jk.3 


Beef kidney 


(1 


81 


77-2 


1.1.5 


73.0 


Beef tongue 


It 


82 


72.2 


k2.k 


h9.k 


Pork kidney 


1! 


83 


78.1 


15.2 


73.0 


Beef kidney 


V 


8h 


79.2 


9-3 


75-6 


Beef kidney 


Stewed 


85 


-- 


10.0 


74. k 


Pork liver 


Dried 


85 


67-8 


9.7 


5^.1 


Beef heart 


Steved 


87 


70.0 


9.0 


80.1 


Beef spleen 


tt 


88 


U3.2 


7.0 


73.8 


Pork loin (frozen) 


Dried 


89 • 


66.6 


33. k 


59-5 


Pork loin (shoulder) 


tt 


90 


71.1+ 


9.7 


31.6 


Pork loin (loin end) 


v 


91 


70.0 


2k. k 


63-5 


Pork loin 


tt 


92 


70.1 


3k. 6 


56.0 


Pork loin 


Fried 


93 


67-3 


32.6 


59.5 


Pork loin 


Roasted 


9h 


7^.0 


25.8 


58. 5 


Pork ham 


Dried 


95 


75-5 


11.8 


75.1 


Boiled ham 


i» 


101 


67.1 


12.2 


61.6 


Tender ham 


tt 


102 


71.5 


11.6 


59.3 


Lamb liver 


m 


96 


71.0 


17.O 


68.7 


Veal liver 


»i 


97 


71.0 


15. k 


6k.-j 


Beef liver 


»i 


93 


-- 


18.9 


6k. 1 


Beef liver 


Fried 


99 


-- 


17.0 


61.0 


Veal leg 


Dried 


103 


73.1 


h.3 


80.7 


Pork heart 


V 


104 


77-7 


9.8 


79.5 



66 



TABLE 71 (continued) 













Per cent Fat 


<fa Protein 












(ether ext. ) 


(Nx6.25) 








Meat 


Per cent 


in dry 


in dry 




Meat 


Process 


sample 


moisture 


tissue 


tissue 


Beef 


round 


Pried 


105 


72.3 


12.8 


71.6 


Beef 


round 


Fried 


106 


— 


22. k 


66.8 


Beef 


round 


Roasted 


107 


78.6 


19.0 


70.0 


Beef 


spleen 


Dried 


108 


-- 


' 8.3 . 


69.!+ 


Beef 


spleen 


Stewed 


109 


-- 


8.3 


72.8 


Beef 


liver 


Dried 


110 


70. k 


13.9 


66.1 


Beef 


liver 


Fried 


in 


56.0 


17.0 


67.2 


Beef 


kidney 


Steved 


112 


6h. 5 


12.2 


73-2 


Beef 


pancreas 


Dried 


113 


73-9 


2^.0 


60. 5 


Pork 


ham 


V 


ill+ 


72.9 


I9.7 


67.0 


Boiled ham 


11 


115 


65.^ 


15-7 


62.1+ 


Smoked ham 


M 


116 


70.6 


15.4 


57.9 


Tenderized ham 


It 


117 


65.6 


' lk.1 


58.1+ 


Pork 


ham 


«' 


118 


72.7 


20.3 


68.6 


Tenderized ham 


ii 


119 


67.0 


17.7 


60.0 


Beef 


liver 


11 


120 


70. k 


-- 


61.6 


Beef 


liver 


Fried 


121 


57-2 


9.1+ 


66.8 


Pork 


liver 


Dried 


122 


68.1+ 


9.2 


59.6 


Beef 


spleen 


ti 


123 


76.1+ 


-- 


76.5 


Pork 


ham (storage) 


V 


12k 


76.1+ 


10.1+ 


78.5 


Pork 


loin 


1! 


125 


72.5 


20.7 


68.6 


Beef 


kidney 


TI 


126 


80.0 


8.8 


72.6 


Beef 


spleen 


<T 


127 


7^.5 


-- 


-- 


Beef 


round 


n 


128 


76.5 


6.2 


79-3 


7eal 


muscle 


1! 


129 


75.2 


6.9 


78.0 


Beef 


spleen 


M 


130 


79.0 


8.6 


7^.6 


Beef 


liver 


It 


131 


70.8 


10.8 


62.9 


Beef 


brain 


M 


132 


78.3 


^8.7 


1+2.5 


Beef 


heart 


V, 


133 


80.8 


11.3 


75-8 


7eal 


hindquarter 


(i 


13k 


78.3 


5. 4 


79.4 


Fillet of cod 


it 


135 


82.9 


1.9 


83.1 


Salmon steak 


tt 


136 


72.2 


20.9 


65-7 



TABLE VI (continued) 



67 



Meat 


Process 


Meat 
sample 


Per cent 
moisture 


Per cent Fat 
(ether ext. ) 

in dry 

tissue 


$ Protein 
(Nx 6.25) 
in dry 
tissue 


Pork kidney 


Dried 


137 


79-9 


12.5 


70.8 


Beef lung 


n 


138 


79-7 


11.0 


75.3 


Pork ham 


i» 


139 


7^.7 


19.6 


70. ij- 


Pork ham 


Fried 


Ha 


k-j.6 


21.7 


69.0 


Pork ham 


Eoasted 


142 


53.O 


2^.7 


55.7 


Beef heart 


Dried 


lk3 


81.0 






Beef "brain 


n 


ikk 


78.75 






Beef round. 


it 


1U5 


74.5 






Beef liver 


it 


150 


72.5 






Beef round- 


»i 


151 


75.2 






Beef liver 


ti 


152 


70.5 






Pork liver 


it 


153 


69.0 






Beef muscle 


it 


154 


75-3 






Veal hearts 


I! 


155 


79.0 






Perk loin 


It 


156 


70.0 






Beef round 


• 1 


157 


72.0 






Veal liver 


It 


158 


73.0 






Pork liver 


1' 


159 


72.0 







69 
LITERATURE CITED 

1. Official and Tentative Methods of Analysis of the Assoc, of Official Agric. 
Chemists, XVII, 22, p. 355 (19^0 ). 

2. Official and Tentative Methods of Analysis of the Assoc, of Official Agric. 
Chemists, XXVII, 3, P . 353 (19^0). 

3. Official and Tentative Methods of Analysis of the Assoc, of Official Agric. 
Chemists, II, 23, p. 26 (19^0). 

k. Haecker, T L. Investigations in Beef Production. I. The Composition of 

Steers at Various Stages of Growth and Fattening. Minn. Agr. Exp. Sta. Bull., 
193 (1920). 

5. Jordan, W 5. The Relation of Food to the Growth and Composition of the Bodies 
of Steers. Maine Agr. Exp. Sta., Part II, 36 (1895). 

5. Konig, J. Die Menschlichen Nahrungs and Gen'ussmittel, ii p. 110. Third 
Edition, Julius Springer, (1893). 

7. Laves, J.B. and Gilbert, J H. Experimental Inquiry into the Composition of 
Some of the Animals Fed and Slaughtered a3 Human Food. Phil. Trans. Royal 
Society of London, Part II, ^93 (1859). 

8. Mrulton, C.R., Trowbridge, P.F. and Haigh, L.D. Studies in Animal Nutrition. 
I, II, III, Changes in Chemical Composition on Different Planes of Nutrition. 
Mo. Agr. Exp. Sta. Res. Bull. 28, 30, 38 and 55 (.1913-1922). 

9. Soxhlet, F. Versuche fiber die Fettbiidung in Tierkorper, Bieder. CentraTbl. 
Agr. Chem., 10, Glh (l88l). 

10. Tcscani, V.A., Rupp, V.R. and McClellan, W.S. Analyses of Meats. J. Nutr., 
k, V73 (193M. 

1' 

11. Tschirwinsky, N. Zur Frage liber die Fettbiidung in Tierschen Organismus. 

Landw. Vers. Sta., 29, 317 (l83l). 

12. Washburn, P.M. and Jones, C.E. Studies of the Values of Different Grades of 
Milk in Infant Feeding. Vermont Agr. Exp. Sta. Bull. 195 (1916). 

11 11 

13. Weiske, H. and Wildt, E. Untersuchungen Uber Fettbiidung in Tierkorper. 

Zeit. f. Biol., 10, 1 (187*0. 

Ik. Wilson, M.E. On the Growth of Suckling Pigs Fed on a Diet of Skimmed Cow's 
Milk. Am. J Physiol., 3, 197 (1902). 

15. Winton, A.L. and Winton, K.B. The Structure and Composition of Foods, Vol. 
Ill, (1937). John Wiley and Sons, Inc., New York. 






_. 






CHAPTER IX 

THIAMIN (VITAMIN B-J 

Page 
Chemistry of Thiamin 71 

Physiology of Thiamin 71 

Pathology of Thiamin Deficiency 73 

Therapeutics of Thiamin 73 

Methods of Assay For Vitamin B-, 7^ 

Rat Grovth Method for Estimating Thiamin in Meats 77 

The Experimental Diet 78 

Results 79 

The Effect of Household Cooking and of Commercial 
Processing upon the Thiamin Content of Animal 
Tissues . 82 



Influence of Fat on Vitamin B-, Results 

Discussion on the Vitamin B-, Assays 

Literature Cited 



81+ 
85 






HIBH 



CHAPTER IX 

THIAMIN (VITAMIN F^) 

Chemistry of Thiamin 

Vitamin 'B was the name given to the original water soluble vitamin, to dif- 
ferentiate it from the fat soluble vitamin A. The gradual increase in knowledge 
over a period of years concerning the water soluble vitamins necessitated the 
division of the so-called B vitamin into a number of factors. A3 more and more 
evidence became available on the multiple nature of the water-soluble vitamin B, 
it became apparent that differentiation could be made between a heat stable and a 
heat labile factor. It was found that the heat labile factor could cure experi- 
mental polyneuritis in chicks and rats and beriberi in humans. Vitamin B, was 
early recognized as the anti -beriberi factor and was considered to be the substance 
which would cure the typical symptoms in humans consuming inadequate diets. This 
substance occurred in appreciable quantities in rice polishings. Funk found (29) 
that acid hydrolysis did not affect the activity of the vitamin, and that fraction- 
ation procedures for the active principle could be performed by taking advantage 
of this property. A. number of investigators, therefore, began work upon the iso- 
lation of the anti -beriberi vitamin from rice polishings. The isolation of the 
vitamin in crystalline form in 193** (99) enabled the complete characterization of 
the vitamin. 

The synthesis of thiamin was accomplished in 1936 both in Europe and in this 
country. The outstanding workers in Europe were Jansen and Donath (39) and Todd 
and Bergel (8k). In Japan the investigators were Ohdake and his co-workers (55). 
The American investigators were led by R. R. Williams and his group (9k } 96,99), 
who contributed largely to the final elucidation of the structure and synthesis 
of crystalline thiamin. This great step forward allowed a greater scope in re- 
searches concerned with physiology, pathololgy, and therapeutics of the many 
clinical manifestations of thiamin deficiency. 



N: 



.C -NH 2 HC1 



CH^— C 



N C— H 



■CH<- 



•CH3 



x C CH 2 CH 2 0H 



Physiology of Thiamin 



Thiamin Chloride Hydrochloride 



The majority of the physiological in vivo and in vitro studies have shown 
that thiamin ia specifically concerned in carbohydrate metabolism (28,57). The 
mechanism of action of thiamin has been related to the breakdown of pyruvic acid 
which i3 one of the intermediary breakdown products of carbohydrate metabolsim 
in the body. The simple decarboxylation of pyruvic acid to acetaldehyde and car- 
bon dioxide in yeast by means of a carboxylase necessitates the presence of a co- 
enzyme, cocarboxylase. This coenzyme ha3 been found to be the diphosphoric acid 
ester of thiamin (50). Thiamin has a similar function in animal tissues although 



-71- 



72 

here the pyruvic acid is not directly deCarhoxylated to acetaldehyde but rather 
oxidatively decarboxylated . The work of.Bahkin (6) has indicated that there is 
some relation of thiamin to the nervous mechanism controlling gastric secretion. 
Molitor and Sampson (53) have tested the influence of pure vitamin B^ on intestin- 
al motility, us^'ng isolated rabbit's intestine suspended in Ringer's solution as 
well as taking observations of the intestine i n situ . These workers found that 
intravenous injection in the in situ experiments or the addition of the pure 
vitamin to the Ringer's solution was without effect on the movements of the in- 
testine. Vitamin B]_ can be absorbed frcm both the large and small intestine, but 
it is to be expected that there will be individual variation in the rate and ex- 
tent of absorption. With this in mind, the question of the occurrence of vitamin 
Bj in the feces attracted the attention of some workers. The synthesis of the 
various B factors by the bacteria in the intestinal tract ha3 been suggested by 
several groups of workers, most notably by Bechdel, Honeywell, Dutcher and Knutson 
(10) and McElroy and Go3s (51). Although this wor v has been done with cows, 
Guerrant, Dutcher and Tomey (32,33) have demonstrated that the type of carbohydrate 
plays an important part in the synthesis of the B vitamins in the rat. 

Anorexia is one of the typical indications of a vitamin B, deficiency in most 
animals. The physiology of anorexia has been placed on a more firm basis by the 
work of Chatterjee (l6) who studied the amplitude, number, and intestinal contrac- 
tions in both vitamin B-, deficient animals and in those getting sufficient B]_. In 
the vitamin deficient animals a definite decrease in physiological response w~a3 
noted for each of the motor tests. 

The effect of avitaminosis B-, on the heart has received the attention of a 
number of workers. A summary of experimental beriberi and the symptomatology ob- 
served in various species has been written by Shimazono (7^+). Vitamin B n also 
plays some role in the normal metabolism of the intact nerve. 

There is abundant evidence that fat an. "spare" the quantity of vitamin B- 
required for normal growth. That is, as the fat content of the diet is increased 
less thiamin is required. It has been suggested by the work of Whipple and Church 
(92) that vitamin Bi plays -a role in the formation of fat from carbohydrate. 
These workers found that animals on a thiamin low diet have a decreased fat re- 
serve while the reserve is increased when the vitamin is added. The use of the 
respiratory quotient to determine the fate of the carbohydrate received attention 
from Whipple and Church (9?). Schradei' (69) has also observed the respiratory 
quotients in vitamin B]_ low rats receiving a carbohydrate diet and found that the 
respiratory quotients averaged 1.26. This may not be conclusive proof but it cer- 
tainly suggests that the conversion of carbohydrate to fat is caking place. It 
is a common practice in the raising of geese to feed or force feed carbohydrate 
in the attempt to fatten these birds. The feeding of a high carbohydrate diet to ' 
pigs in order to fatten them is a similar illu3tra + ion. It is of interest, how- 
ever, to note the work of Eemmerer and Steenbock (^3) who found a lower vitamin 
3t_ content of the muscle of a pig fed ^0 per cent lard than in the muscle of the 
normally fed pig. It would appear that the higher content of thiamin in pork 
muscle is an indication of the need of thiamin for the conversion of carbohydrate 
to fat. The more carbohydrate fed, the greater is the need for thiamin, and since 
thiamin functions in some manner in the conversion of carbohydrate to fat, it would 
be expected that more thiamin would be mobilized into the muscle of the carbohy- 
drate fed animal than in the fat fed animal . 



73 



Pathology of Thiamin Deficiency 



Although the condition of beriberi is primarily a disease of the Orient, the 
recognition of subclinical deficiencies due to a lack of this vitamin is becoming 
more apparent as disclosed by perusal of the recent medical literature. The 
diagnosis of polyneuritis has been made in cases of the chronic alcohol addict, 
in certain pregnancies, in complicated anemias, in cardiovascular disfunction, in 
epigastric distress, and in many other clinical conditions (86). 

One of the striking symptoms of a thiamin deficiency is that of the "beri- 
beri heart" in which there is marked hypertrophy and dilatation of the right side 
of the heart. There is a resultant edema, cardiac failure, and circulatory col- 
lapse. Wenckebach (90) and Aalsmeer and Wenckebach (l) have given a partial ex- 
planation for these symptoms by suggesting that the enlargement of the heart is 
actually due to an edema. Weiss and Wilkins (89) also intimated that edema played 
a part in the cardiovascular pathology. 

There has always been a tendency to associate a vitamin B-, deficiency with 
certain changes in the nerve structure. The paralysis resulting from avitaminosis 
B]_ has been linked with changes, in the spinal cord. Bentley (ll) has reported the 
results of 19 necropsies and came to the conclusion that the greatest pathology 
occurred in the spinal cord. Wright (100) has also studied this question and has 
found changes in the anterior horn cells and in the spinal ganglia. Myelin sheath 
degeneration of peripheral nerves has been described by several workers. Engel 
and Phillips (23) have reported histopathological findings in vitamin B-, deficient 
chicks and rats. These workers claimed that the administration of adequate 
^-carotene, riboflavin and sufficient vitamin D removed all evidence of pathology 
in the peripheral nerves of the chick and rat. They concluded that there is no 
neuropathology in the uncomplicated vitamin Bi deficiency of these animals. 
Prickett (63) drew the surprising conclusion that the site of the lesion respon- 
sible for the symptoms of vitamin B, deficiency is in the central rather than in 
the peripheral nervous system. Gr inker and Kandel (31 ) have presented evidence 
that there is no nervous system degeneration in experimental thiamin deficiency. 
The apparent disagreement on the subject has been correlated by Street, Zimmerman, 
Cowgill, Eoff and Fox (83). These workers suggest that the most probable reason 
for the variation in results is due to the fact that several of the deficiencies 
reported were not "complete vitamin Bj deficiencies." This group also state that 
a moderate shortage of the vitamin over an extended period may produce pathology 
which is different from that seen in the acute disease. 

Therapeutics of Thiamin 

The availability of crystalline thiamin has made it possible to utilize this 
vitamin for clinical conditions which demand immediate treatment. One such condi- 
tion is acute polyneuritis whose sudden onset is due to gastric difficulties. 
Cardiovascular involvements resulting from a sudden deficiency of thiamin has been 
treated with vitamin B-j_ (8l). There is come evidence supporting the use of thia- 
min in complicated cases of malnutrition, such as in chronic pellagra. The recog- 
nition of polyneuritis of pregnancy as a dietary deficiency has resulted in the 
relief of patients who otherwise would have been treated for toxemias (80,82). A 
polyneuritis attributed to lack of thiamin in alcohol addicts has been referred to 
by a number of investigators (40) . 



The medical literature is replete with studies on the subclinical conditions 
relating to thiamin deficiency. An editorial in the Journal of the American Medi- 
cal Association points out the need for the adequate intake of this important 
vitamin and the statement is made that "the slim margin that exists between man's 



7^ 

physiologic requirements for vitamin B-j_ and his intake of this vitamin has long 
teen known; also the fact that storage of this vitamin is minimal and that debil- 
ity quickly follows when men are deprived of it." Some recent work (97) has "been 
reported on the induced vitamin Bn deficiency in human patients. When normal 
human subjects were restricted in their intake of thiamin but otherwise receiving 
an adequate diet, they showed signs of sluggishness, moodiness, indifference, fear 
and mental and physical fatigue. When other subjects on the same experiment re- 
ceived thiamin they remained in apparent good health for a number of months. The 
interesting observation was made that when the level of thiamin was raised to 600 
international units per day, the subjects showed an enlargement in the capacity 
for physical work as well as an increase in alterness. The usefulness of thiamin 
administration in a variety of neuritic states is summarized in the chapter devoted 
to that subject by Williams and Spies (?8). A large number of papers has dealt 
with borderline cases of thiamin deficiency in conditions of beriberi heart, . 
alcoholic polyneuritis j polyneuritis of pregnancy, hyperthyroidism, diarrhea, 
anorexia, severe pellagra, Korsakoff's syndrome, post irradiation nausea, sprue, 
multiple sclerosis, sciatica, rheumatoid arthritis, sacro-iliac arthritis, and 
nerve deafness. Although some of these conditions cannot be attributed to a clear- 
cut thiamin deficiency, it is becoming increasingly apparent that the beneficial 
effects of the administration of the vitamin can be attributed to the co -existing 
nutritional deficiency. 

M ethods of Assay for Vitamin v3 j 

Since thiamin is the oldest of the water soluble vitamins, it is to be expect- 
ed that numerous methods would be proposed for the- determination of this vitamin. 
Any summary of the available methods at the present writing must consider all the 
published proposals, but at the same time give due recognition to the more reli- 
able ones. This neceaaltates a critical appraisal of these methods. An important 
consideration deals with the question of the completeness of the diet used in the 
biological procedures. There are a number of new factors which must be included 
in experimental diets in order to achieve the greatest accuracy of the bioassay. 
Another point to be kept in mind is whether the values derived by these methods 
were obtained by comparison with a reliable standard such as an international 
fullers earth adsorbate or with crystalline thiamin. The discussion of the thia- 
min values in the literature will enlarge on this no int. 

Many of the biological methods can be classified according to the type of 
assay. There are methods which depend solely upon the growth response obtained 
with the vitamin or with supplements containing the vitamin. Methods have been 
proposed which have been based upon the prevention of polyneuritis, on the ability 
of a particular supplement to maintain the weight of adult individuals, and on the 
cure of the typical polyneuritis in various animals. Less reliable methods depend 
upon the ability of the foodstuff to restore appetite or .to cure particular symp- 
toms-such as nystagmus. The choice of a method is dependent upon the potency of 
the supplement, the number of samples to be assayed,- the speed, and the accuracy 
desired. Recent attempts have been made to obtain uniformity in the chemical de- 
terminations, especially for the thiochrome procedure. A 3.hort review of the 
available biological and chemical methods is given below. 

The original work on beriberi in fowls was done by Grijns and Eijkman. Later 
polyneuritis waa also produced in chickens. Several laboratories have contributed 
methods based upon the cure or prevention of polyneuritis in pigeons (22,^-6). 
Weight maintenance methods using pigeons have also been used ( 13,61,72). 

A method which utilizes chicks as the experimental animal has furnished re- 
liable values for certain materials '(5). An autoclaved ration containing yellow 
corn, wheat middlings and casein, and supplemented with all the known factors 



75 

except thiamin is fed to day-old chicks. The growth response obtained on the 
material to "be assayed is compared with the growth obtained when crystalline thia- 
min is added to the ration. Some of the values which are listed in the table for 
the vitamin Bi content of meats were checked by the chick method. 

A method involving the uptake of oxygen of a pigeon's brain was used for 
3ome time. It depended upon the increased oxygen uptake of the polyneuritic 
pigeon's brain when concentrates of the vitamin were added to the brain suspen- 
sion. The use of a reference curve, which related the amount of vitamin added 
to the quantity of oxygen consumed, enabled the investigator to calculate the B^ 
in the supplement. This catatorulin te3t (56) has now outlived its usefulness. 

The use of rats as the test animal perhaps attracted the attention of most 
of the early workers in the vitamin B-i field. The Sherman and Spohn diet (73) was 
used for several years for the experimental production of polyneuritis in rats. 
This was modified by Chase and Sherman (15) so as to dupply the heat stable factor 
shown to be required by rats. The variation in the diets has been one important 
factor in the modifications proposed, but the technique has furnished the means 
by which a curve can be constructed plotting the growth responses against the 
amounts of vitamin Et used. 

The development of diets low in vitamin Bj_ has also enabled the use of cura- 
tive procedures for the estimation of the vitamin. The procedure was first pro- 
posed by Smith (75). Hofmeister (37) used large numbers of rats which had poly- 
neuritis and when Kinnersley, Peters, and Reader (hj) compared the curative activ- 
ity of Bj_ upon pigeon and rat polyneuritis essentially the same values were ob- 
tained. This was adequate proof that both methods' were measuring the 'same thing. 
The rat curative procedure and technic is essentially the same regardless of the 
diet that i3 used. The official U. S. Pharmacopeia XI, 2nd supplement, lists the 
ration that is used in the curative tests. The cure of polyneuritic rats with 
small amounts of crystalline vitamin is compared with the duration of cure exhibit- 
ed by the same rats which receive the vitamin supplement after polyneuritis is 
again induced. By adequate standardization the same rats can be used for a number 
of assays (48) . 

A new synthetic thiamin low diet has been developed by Waisman, Henderson and 
Elvehjem (88) using a simplified diet which contains a: 1 the known factors neces- 
sary for the rat. The new ration can be used either for prophylactic assays or 
for curative procedures. Waisman and co -workers have obtained data which sub- 
stantiate the claim that. the duration of cure of polyneuritis in rats is propor- 
tional to the levels of thiamin administered when .levels up to 10 micrograms are 
used. There is a direct relationship .also to the gain in weight with different 
levels of thiamin either fed or injected. 

Drury, Harris and Maudsley (20) developed a method for estimating vitamin B-, 
based upon the bradycardia associated with vitamin 3-, deficiency. The electro- 
cardiogram has been used to refine the bradycardia method by furnishing an accurate 
estimation of the heart rate. Birch and Harris (12) have given reliable figures 
for a variety of foods using the special electrocardiogram. The advantage of 
economy in test animals, rapidity and ease of method are offset by the high initial 
cost of the instrument. 

The use of microorganisms for estimating thiamin has been based on the linear 
response in growth of the organisms with known levels of the vitamin. Bacterio- 
logical media containing ail the known factors necessary for growth of a particu- 
lar organism except thiamin are used for the test. The method developed by West 
and Wilson (91) uses Staphylococcus aureus as the test organism. The growth of 
the organism is plotted against the amounts of the added vitamin giving the 



76 

standard curve. Comparisons of the growth on the test supplements to that of the 
actual quantity of thiamin can he obtained. The growth is determined by means of 
turbidity measurements using a. suitable photoelectric colorimeter or turbidometer. 
Schopfer and Jung (60) have reviewed the possibilities of using the mold Phycomy- 
ces blakesleanu s for vitamin B- assays. 

One of the most recently proposed methods is the fermentation test described 
by Schultz, Atkin and Frey (70). They have described the method in detail to- 
gether with the precuations necessary for obtaining good values. The quantitative 
method involves the measurement of the rate of gas evolution. The greatest ob- 
stacle in the use of this method is the preparation of a medium which is free of 
the vitamin, but complete enough to give optimum growth of the organism. The in- 
fluence of various pyrimi dines, obtained from thiamin decomposition, upon the rate 
and extent of fermentation ha3 been investigated by these workers. Schultz, At kin 
and Frey (70) claimed that good results are obtained by this procedure if a blank 
correction is applied for the presence of stimulating pyrimi dines not derived from 
thiamin. Recently, Schultz, Atkin, Frey and Williams (71) have applied the sul- 
fite cleavage of thiamin to the yeast fermentation method, and have found that a 
true thiamin content of a substance can be obtained by the differences in fermen- 
tation before and after sulfite cleavage. 

The biological assay methods used for following the activity of the various 
fractionation procedures have served an important purpose, but often the bioassay 
methods are displaced ^>j chemical tests when the structure of the vitamin is known. 
This is perhaps as it should be for the isolation and synthesis of a new factor 
enables greater precision in the method used in its determination. This in no 
way detracts from the usefulness of bioassays, but rather serves to augment the 
rate of progress by furnishing preliminary information as well as fairly reliable 
preliminary values . 

In 193? Peters (53) reported that one of the properties of his vitamin Bt_ 
preparations was the susceptibility to oxidation to a bluish fluorescent compound. 
This oxidation was accomplished by Barger, Bergel and Todd (9) by the use of alka- 
line ferri cyanide. They were quick to realize that the blue fluorescence in 
neutral and alkaline solution had all the properties of thiochrome described pre- 
viously by Kuan, Wagner -Jaur egg, Van Klaveren and Vetter (^9). Several other 
workers then attempted to use the conversion of thiamin to thiochrome as a means 
of quantitatively determining the vitamin. Jans en (33), Goudsmit and Westenbrink 
(30), fyke (6k) } and Karrer and Kubli (kl) described their procedures for this de- 
termination . 

It was soon apparent that some interfering material was imparting fluorescence 
that did not have vitamin B-i activity. Hennessey and Cerecedo (36) employed zeo- 
lites for absorbing out the vitamin. Several refinements in the method have ap- 
peared (35) • 

A color reaction dependent upon the specificity of the thioazole nucleus in 
reacting with para amino acetanilid or methyl para amino phenyl ketone in the 
presence of nitrous acid has been the contribution of _ Prebluda and McCollum (62). 
Melnick and Field (52) have modified the method and have applied it to determina- 
tion of urine and blood thiamin. Raybin (66) has reported that thiamin will react 
with 2,6 dibromoquinone chloroimide to give an orange compound which is readily 
extractable by immiscible solvents. Naimahs (5^) also got an orange red precipi- 
tate when bismuth potassium iodide was added to a solution containing vitamin Bj_. 
.Another color reaction reported by Jansen and Donath (39) was that resulting from 
the interaction of diazotized sulfanilic acid with sodium carbonate in the presence 
of vitamin B-i.. This reaction was later investigated by Kinnersley and Peters (k^>) . 






77 

The difficulties involved in using any of the chemical methods for the deter- 
mination of thiamin are those inherent in any chemical approach, namely, that 
there are a number of interfering substances which may upset the expected chemical 
reaction thus making it nonspecific. While the chemical methods work admirably 
on pure solutions of the vitamin, the widespread use of extraction or absorption 
procedures always entails the probable loss of the vitamin. Some methods will 
work well with urine, others with blood, but the desired ready application of 
most methods to a natural foodstuff has not yet been obtained. Although there 
is good reason to believe that the chemical methods hold promise in this direction, 
further improvements are necessary. 



Pat Growth Method for Estimating Thiamin in Meats 

It is apparent from the discussion on the available methods for estimating 
thiamin, that each approach has something to be desired. The rat growth method 
is perhaps the most reliable indication of the ability of a certain supplement to 
prevent the typical symptoms of a vitamin B-, deficiency. A bioassay furnishes a 
true measure of the physiological response of a particular supplement and can be 
best compared with the physiological response obtained with the pure vitamin. The 
limitations of the method are mainly dependent upon the basal diet used. It is 
only necessary to point out that the basal ration should be complete in all the 
known crystalline vitamins together with purified concentrates of established fac- 
tors which are suitably free of thiamin. The use of an adequate diet and standard- 
ized rats appears to be the most reasonable -procedure for the bioassay of vitamin 

B l- 

The rat growth method employed for the systematic determination of thiamin in 
muscle and organ tissue is that of Arnold and Elvehjem (h) . The diet used is simi- 
lar to that which has now been accepted as the official diet and is described in 
the U. S. Pharmacopeia, 2nd supplement (35). The assays to be reported here were 
performed by the prophylactic technic, while the U. S. Pharmacopeia procedure in- 
volves the curative approach, developed by Kline, Tolle, and Kelson (U8) . 

All the rats used in the assays were obtained from a reliable commercial 
source, thus eliminating as much as possible individual variation and genetic dif- 
ferences. The twenty-one day old albino male and ^emale rats were kept on the 
basal ration for one week so as to further equalize the differences that may have 
existed within the litters. In the later experiments only male rats were used in. 
each series of assays of forty or more animals . Usually four or more animals were 
used for each level of meat in one or more series. The rats were placed in indi- 
vidual screen bottomed cages and fed the diet and water ad libitum. Both positive 
and negative controls were part of each series, as well as the several groups of 
rats which received various levels of the standard vitamin solution. The crystal- 
line vitamin B, hydrochloride was made up so that 100 micrograms were dissolved in 
1 milliliter of tenth normal acid. This standard was added to some of the basal 
ration and carefully dried in a vacuum desiccator at room temperature. After dry- 
ing, the small amount of ration was mixed with sufficient basal ration to give the 
concentration desired. One group received Uo micrograms per 100 grams ration, 
another 60, another 80, and usually one at 100 micrograms. Occasionally a group 
receiving 120 micrograms was also included. After a number of series were run, it 
became apparent that the 80 microgram level furnished the growth that could best 
be used as a basis for the computation of the vitamin B-i content. In the later 
series, therefore, only the 80 microgram level was fed. In all our series, crys- 
talline vitamin B]_ was used. Knowing the level of meat which produced growth 
equal to that of pure thiamin, the vitamin content of the meat could be calculated 
by means of a simple proportion. The acceptance by the permanent commission on 
biological standardization of the Health Organization of the Lea.gue of Nations that 



78 

3 micrograms of thiamin are equivalent to one international unit allows us to 
express our results both in micrograms and international units per gram of the 
fresh and dried tissue. 



The meat supplements were added to the basal ration at various levels . These 
levels were so adjusted that they produced a gain in weight comparable to that ob- 
tained on the standard levels of crystalline thiamin in that particular series 
of assays. The period of assay was six weeks, but the condition of the rats was 
noted carefully throughout the experiment. The animals were weighed weekly and 
the growth was charted and compared to the growth of those rats receiving the pure 
vitamin. 

The Experimental Diet 

The ingredients and composition of the experimental vitamin B - low ration 
is as follows: 



Sucrose 






62 


Purified casein 






18 


Autoclave! peanuts 






10 


yeast 






k 


Salts 1 






k 


Purified liver frac 


tion 




2 


Halibut liver oil 




2 


drops/wk 



The sucrose is the ordinary commercial cane sugar. 

The casein is purified by dissolving commercial casein in dilute NHhOH and 
diluting the solution before the addition of hydrochloric acid to precipitate the 
casein. The whey is decanted and the precipitate is washed and again dissolved 
in alkali. This washing and precipitation is alternately done 5 times. The puri- 
fied casein is then dried in shallow pans and finely ground. 

Unroasted shelled No. 1 grade peanuts are crushed in a meat grinder, auto- 
claved for five hours at 12C°C. at 15 pounds steam pressure. The peanuts are 
spread in pan3 in layers not exceeding 12 mm. in depth. The peanuts are then 
dried in a current of warm air for lo hours. 

The autoclave! yeast is prepared in a manner similar to that of peanuts. 

The salts can be any complete salt mixture. 

The purified liver extract is prepared by dissolving 100 gms. of liver ex- 
tract (pernicious anemia preparation) 1:20 in 200 cc . of distilled water. A liter 
of alcohol is added together with 1200 cc. of ether (ethyl). The mixture is well 
shaken in a 3 gallon glass flask or bottle. After precipitation is complete, the 
supernatant mixture of alcohol and ether is poured off, and the residue is dis- 
solved in 200 cc. of water and again one liter of alcohol is added with another 
1200 cc. portion of ethyl ether. This is repeated once more and the precipitate 
dissolved to a known volume, preferably 100 cc . 

The liver preparation is spread on the sucrose and dried at 38°C. before a 
fan. The remainder of the ingredients are added and the entire mixture is ground 
in a burr mill. 



79 

This ration and the meat supplemented rations were kept in a cold room be- 
tween feedings. The feedings of halibut liver oil bo each rat avoided the neces- 
sity of adding a source of vitamins A or D directly to the ration. In no case was 
any rancidity observed in the basal ration or . in the meat supplemented ration. 

Pesults 

The preparation of our meat samples has been described previously, but before 
the method of drying was adopted, it was necessary to obtain information as to the 
stability of the vitamins during the drying process. To determine if any thiamin 
was destroyed in the tissues, both the fresh meat and the dried meat were assayed 
simultaneously in the same experimental series. In one trial four groups of de- 
pleted rats were fed the basal ration plus 1, 2, 3 and 6 per cent, respectively, 
of the dried pork ham (sample 32).. Daily food consumption records were made for 
all rats on the dried meat. In another group within the same series, the rats 
received the fresh pork ham in daily supplements equivalent to the dry meat con- 
sumed by the rats in the corresponding group . Those rats receiving the fresh meat 
supplements had free access to the basal ration at all times. By calculating the 
two results, it was found that there was very little difference in the two assays 
of the same sample. In a similar series of assays with pork loin, it was found 
that both the dried and the fresh tissue contained 13 international units of the 
vitamin per gram dry tissue. A similar set of experiments was carried out with 
pork liver, and here it was shown that a value of 5-3 international units per gram 
dry weight basis was obtained by both methods. These results warrant the conclu- 
sion that very little destruction, if any, occurs during our method of drying. 

Our extensive assays demonstrated that there is an exceedingly high amount 
of thiamin in the muscle of pork. Although samples of both ham and loin varied 
in their vitamin Bj_ content all contained large amounts of the vitamin. Six dif- 
ferent samples of pork ham showed a range from 33 to 73 micrograms of thiamin per 
gram of the dried sample. When eight samples of p: rk loin were assayed, it was 
found that three of the samples contained 39 micrograms per gram, while five others 
showed a range of from k-8 to 78 micrograms per gram. These values are an indica- 
tion of the relatively high content of thiamin in pork muscle and the range one 
can expect in a number of such samples. In order to verify these high values 
some of the samples of pork were tested by the chick method (5). The results ob- 
tained by this method gives values which compare well with those obtained by the 
rat assay procedure. A further check on the high values of pork was obtained by 
the microbiological method described in a previous section (91). Here again, there 
was good agreement by the two methods. 

The thiamin content of pork muscle is considerably higher than the muscle of 
other species. Lamb and veal muscles contain slightly more thiamin than beef 
muscle. With the exception of one veal hindquarter the samples contained approxi- 
mately one-third to one -fourth the thiamin content of pork muscle. 

The thiamin content of the various livers was fairly constant in contrast to 
the rather wide species variability of the muscular tissue. The samples of beef 
liver, veal liver and lamb liver showed values in about the same range as the 
muscular tissue of beef and veal. 



Other animal tissues showed a wide variation in their thiamin content. Beef 
heart and pork heart are very good sources of thiamin together with pork kidney. 
These tissues contain about one-half of that found in pork muscle. Tissues such 
as pancreas, brain, lung, and tongue contain low amounts of thiamin in comparison 
to the more potent tissues being in the order of about one -tenth of the pork ham. 



8o 

Spleen alao contains the same amount of vitamin B-, as the other organs. Several 
chicken samples that were assayed indicated that the dark meat showed a higher 
thiamin content than the light meat. This finding was true for "both the younger 
and older tissues which were assayed. The first dark muscle and the first light 
muscle were obtained from cocks which were nearly three years old. The second 
samples of dark and light tissues was prepared from a pooled lot of 15 pullets 
about 15 months old. 



TABLE VII , 



Meat 



Thiamin (Vitamin B ) Content of Meats 

Mcgm./gm. Mcgm./gm. I.U./gm. I.U./gm 

Sample Meat fresh dry fresh dry 

number process weight weight weight weight 



Eeef 


brain 


57 


Dried 


2.5 


Beef 


heart 


53 


tt 


6.8 


Beef 


heart 


73 


11 


;.s 


Beef 


kidney 


h9 


it 


3.2 


Beef 


kidney 


81 


11 


2.1 


Beef 


liver 


58 


11 


3.9 


Beef 


liver 


93 


11 


— 


Beef 


liver 


120 


11 


3-6 


Beef- 


lung 


63 


it 


2.0 


Beef 


pancreas 


64 


ti 


3-2 


Beef 


round 


1+0 


ti 


2.8 


Beef 


round 


105 


n 


1.7 


Beef 


spleen 


59 


11 


1.6 


3eef 


tongue 


67 


ti 


2.8 


Lamb 


liver 


61 


11 


4.1 


Lamb 


leg 


35 


ti 


2.6 


Lamb 


leg 


55 


H 


3.3 


Veal 


hindquarter 


56 


I! 


1.1 


Veal 


ti 


44 


II 


3.9 


Veal 


n 


75 


II 


3-5 


Veal 


I! 


103 


II 


3.2 


Veal 


liver 


97 


II 


5.2 


Pork 


ham 


32 


ft 


9.1 


Pork 


ham 


32 


Fresh 


9.1 


Pork 


ham 


51 


Dried 


15.3 


Pork 


ham 


95 


11 


19.0 


Pork 


ham 


.114 


11 


12.7 



8.0 

30.0 

18.0 

15.0 

9.0 

12.0 

15.6 

9.0 

9.0 

10.0 

9.0 

6.0 

7.0 

9.0 

12.0 

12.0 

12.0 

5-2 

13.5 
15.0 
12.0 
18.0 
33.0 
30.0 
60.0 
78.0 
47.0 



0.8 
2.1 

1.3 
1.0 

0.7 

1.3 

1.2 

0.7 
1.1 
1.0 

0.6 
0.5 
0.9 

1.4 

0.9 
1.1 

0.4 

1.3 

1.2 
1.1 
1.4 
3-0 
3.0 

5.1 
6,3 

4.2 



2.7 

10.0 

6.0 
5.0 
3.0 

4.0 

5.2 

3-0 

3.0 
3.3 

3.0 
2.0 

2.5 
3.0 
4.0 

4.0 
4.0 

1.6 

U.5 

5-0 
4.0 

6.0 
11.0 
10.0 
20.0 
26.0 
15.7 



TABLE VII (continued) 



81 











Mcgm./gm. 


Mcgm./gm. 


I.U./gm. 


I.U./gm. 






Sample 


Meat 


fresh 


dry 


fresh 


dry 




Meat 


number 


process 


weight 


weight 


weight 


weight 


Pork 


ham 


118 


Tried 


14.2 


52.5 


. 1*.7 


17.5 


Pork 


ham 


124 


t! 


14.9 


6*.0 


5-0 


21.0 


Boil< 


2d ham 


101 


»t 


11.8 


36.O 


3.9 


12.0 


Boiled ham 


115 


II 


11.1* 


33.0 


3.8 


11.0 


3mok; 


3d ham 


kj 


Tl 


10.7 


33.0 


3.5 


11.0 


Smoks 


2d ham 


116 


II 


9.4 


31.8 


3-1 


10.6 


Tender ham 


102 


II 


6.0 


21.0 


2.0 


7.0 


Tender ham 


117 


II 


7.8 


22.5 


2.6 


7.5 


Tender ham 


119 


•1 


8.9 


27.0 


3.0 


9.0 


Canned ham 


72 


II 


8.2 


26.0 


2.7 


8.6 


Pork 


heart 


71 


II 


5-2 


25.5 


1.8 


8.5 


Pork 


heart 


104 


II 


7.0 


31.5 


2.3 


10.5 


Pork 


kidney 


62 


11 


5.2 


24.0 


1.8 


8.0 


Pork 


liver 


33 


II 


1*.7 


16.0 


1.5 


5.3 


Pork 


liver 


33 


Fresh 


4.4 


15.5 


1.4 


5.0 


Pork 


liver 


122 


Bried 


5.7 


18.0 


1.9 


6.0 


Pork 


loin 


3h 


11 


ll.l 


39.3 


3.7 


13.1 


Pork 


loin 


3^ 


Fresh 


ll.l 


39.0 


3.7 


13.0 


Pork 


loin 


36 


Dried 


18.7 


60.0 


6.2 


20.0 


Pork 


loin 


7^ 


11 


16.0 


57.0 


5.3 


19.0 


Pork 


loir- 


89 


11 


26.0 


78.O 


8.7 


26.0 


Pork 


loin 


90 


11 


13.7 


48.0 


4.6 


16.0 


Pork 


loin 


91 


11 


11.7 


39.0 


3.9 


13.0 


Pork 


loin 


92 


11 


11.6 


39.0 


3.9 


13.0 


Pork 


loin 


125 


it 


11*. 7 


54.0 


4.9 


18.0 


Poultry, dark 


66 


11 


2.3 


9.0 


0.9 


3.0 


Poull 


;ry, dark 


69 


ti 


1.8 


7.6 


0.6 


2.5 


Poultry, light 


65 


i< 


1.5 


5.5 


0.5 


. 1.8 


Poultry, light 


63 


11 


0.7 


3.0 


0.2 


1.0 



82 

The Effect of Household C ooking and of Commercial Processing upon the Thiamin 
Content of Animal Tissues 

The results of the thiamin assays indicate that there is destruction of this 
vitamin in most cooking processes. The results are summarized in Table VIII. It 
will be seen that frying produced the least change in antineuritic potency. The 
two samples of beef liver showed 13 and 23 per cent destruction of the .vitamin, 
while the two beef round steak3 differed in the apparent loss. One sample shewed 
no loss, and the other indicated that 30 per cent destruction occurred by our 
method of frying. The remaining muscle meats also varied in the loss of the vita- 
min. One sample of pork ham showed no loss after frying, while a smoked ham and 
pork chops indicated a 12 and 35 P er cent loss respectively. Another set of pork 
chops lost 53 per cent of thiamin. There is at present no explanation for the 
variation in destruction of the vitamin in the various samples. 

It was evident from the cooked samples that roasting caused appreciable de- 
struction of thiamin. The two round samples of beef that were roasted showed 55 
and 56 per cent destruction, and the sample of veal also showed a like amount of 
destruction. It is interesting to note that the sample of pork that was roasted 
also showed a loss of 50 per cent. It is apparent that the greater destruction 
that occurs in the roasting process is possibly associated with the longer cook- 
ing time and with the increased temperature. A single sample of beef round was 
broiled according to the accepted procedures and this sample also showed a loss of 
50 per cent when compared to the same sample before cooking. 

The stewed samples were heart and kidney of beef. The loss in these two 
organs amounted to J +0 per cent for the kidney and 57 per cent for the heart. In 
both cases the losses on cooking in all probability may be explained on the extrac- 
tion of thiamin from the meat by the water in which the tissues were cooked. The 
extent of these lo33es by leaching are usually reduced in the preparation of stews 
since the cooking water is usually incorporated into the final serving. In our 
samples the stew liquid was not assayed. 

At the present time there is no reliable data on the temperatures attained 
inside the meats during the cooking processes and so a correlation cannot be made 
between the stability of the vitamin and the heat to which a particular tissue 
is subjected. The prolonged cooking time usually employed in the roasting of 
meats is evidently of greater importance than the actual temperature since the 
frying produced less loss of the vitamin. Further work is necessary to establish 
this point. 



83 



TABLE VIII 
Stability of Thiamin During Household Cooking Processes 



Meat 



Meat 
process 



Sample 
number 



Time of 
processing 



Thiamin 
content 



Per cent 
destruction 
of thiamin 



Beef liver 


None 


98 


» tt 


Fried 


99 


it it 


None 


120 


H ti 


Fried 


121 


Beef round 


None 


40 


ii it 


Broiled 


41 


it ii 


Fried 


42 


ii n 


Roasted 


^ 


it ti 


None 


105 


it it 


Fried 


106 


n tt 


Roasted 


10? 


Veal hindquarter 


None 


kk 


ti it 


Fried 


45 


tt it 


Roasted 


he 


Pork ham 


None 


51 


it it 


Fried 


52 


Smoked ham 


None 


47 


n it 


Fried 


48 


Pork loin 


None 


36 


it tt 


Baked 


37 


i» it 


Fried 


38 


it ii 


Roasted 


39 


ti tt 


None 


92 


ti tt 


Fried 


93 


it it 


Roasted 


94 


Beef heart 


None 


53 


ti it 


Stewed 


5^ 


Beef kidney 


None 


h9 


ti ii 


Stewed 


50 



15 min. 
15 min. 

20 min. 
11 n 

2-1/2 hr. 

20 min. 
2 hr. 

20 min. 
2 hr. 

15 min. 

15 min. 

1 hr. 
25 min. 
1-1/3 hr, 

25 min. 
105 min. 

60 min. 
45 min. 



mcg./g. 
dry wt. 

15.6 

12.0 
9-0 
7.8 
9.0 

9.0 

4.0 

6.0 

4.2 

3.9 

13.5 

7.5 

6.0 

60 

60 

33.0 
29.0 
60.0 
30.0 
39.0 
30.0 
39.0 
18.0 
24.0 
30.0 
13.0 
15.0 
9.0 



23 

13 

50 



56 

30 
55 

44 

55 



12 

50 

35 
50 

53 
38 

57 

40 



Qk 

Influence of Fat on Vitamin B i Results 

The original concept of the sparing action of fats on the vitamin B-j_ require- 
ment of rats was that of Evans and Lepkovsky (2k-26) who showed that more vitamin 
B-^ is necessary for the growth of rats on sucrose diets than for those animals on 
diets containing some fat substituted for the sucrose. Salmon and Goodman (67) 
have studied the question and reported the cure of polyneuritic rats by feeding 
high levels of natural and synthetic fats. These workers found that the length 
of the carbon chain was of importance in effecting the alleviation of the symptoms 
of polyneuritis. Stirn, Arnold and Elvehjem (79) found no marked difference 
between olive oil, coconut oil, autoclaved peanut oil, autoclaved lard, and auto- 
claved cottonseed oil in alleviating the typical thiamin deficiency symptoms when 
the fats isocalorically replaced the carbohydrate. Synthetic fatty acid esters, 
tricaproin, and triacetin were also effective in relieving the polyneuritis in the 
rats. 

This ability of fats to offset polyneuritis as well as its ability to cure 
spastic polyneuritis when sufficiently high fat was fed, led to the question of 
the probable influence that the fats in our meat samples might have on the results 
of the assays. In order to test this possibility, experiments were begun in which 
various groups of rats were fed the basal ration and the basal ration with increas- 
ing amounts of fats. Each group also received amounts of crystalline vitamin which 
were known to be inadequate for normal growth. It was felt that if the fat had 
any sparing action on the vitamin requirement, then the addition of insufficient 
amounts of the vitamin should give as good growth as optimum levels of thiamin. 
It was found than when 2, k, 6, 8, or 10 grams of coconut oil and 20 micrograms 
of vitamin B, were each added to 100 gram3 of the basal ration, there was no ap- 
preciable difference in the increase in weight of these animals over a six weeks' 
period compared to the increase ma.de by those rats receiving the basal ration plus 
20 micrograms of vitamin B]_ per 100 grams of diet. From data obtained over a long 
period it was apparent that the total increase in the weight of the rats on the 
added fat ration can be attributed solely to the small amount of the vitamin which 
was added. 

In another series 10 percent coconut oil was fed together with 20, ^-0, or o0 
micrograms of vitamin B^; 20 percent coconut oil was fed with 20 and ^0 micrograms; 
and 30 percent coconut oil was fed with 20 micrograms of the vitamin. When the 
level of vitamin Bj_ was increased to kO micrograms per 100 grams of ration and 
the fat left at 10 percent the rats again grew no better than on the 40 micrograms 
alone. The same results were obtained in the remaining run3 which contained 10 
percent coconut oil and 60 micrograms of vitamin Bt. When the level of fat was 
increased to 20 per cent, the growth of the rats was no better with the added 
vitamin than it was in rats receiving the basal ration with the usual amount of 
fat and standard amounts of the vitamin. 

The experiments described above on the fat sparing action on vitamin Bj 
strongly indicate that the amount of fat furnished by the meat supplement was too 
low to influence the results of the assays. Since the tissues were added directly 
to the basal ration and because they were not added at the expense of any one 
constituent, there ia little basis for the criticism that a particular part of 
the diet was favored which might have affected the results. The addition of the 
meat supplement to the ration was usually at a level less than 10 per cent. At 
this low level, even in samples containing as much as 50 per cent fat, our experi- 
ments showed that the per cent of fat added to the ration by the tissue is in 
that range where it has little influence on the growth of the rats maintained on 
this particular ration. We can conclude, therefore, that the thiamin results are 
reliable. 



..» 



85 



Discussion on the Bj Assays 



The progress in investigative "biochemistry during the past few years has re- 
sulted in the demonstration that many of the important accessory food factors can 
he found in foodstuffs previously considered to he devoid of them. One of our 
first attempts in the investigation of the vitamin content of meat was directed 
toward the occurrence of thiamin in meat and meat products since thiamin has "been 
one of the vitamins which has received much attention in the clinic. Since meat 
plays an important role in the human diet, it was helieved worth while to know 
mere about the vitamin B^ content of ordinary cuts of meat and certain meat by- 
products. 

. '■ ■■%?■ 

A striking fact ahout the results of the thiamin content of pork muscle is 
the variation of the vitamin in this particular type of tissue. The range of 
values that one ohtains is not only due to the expected variation in a large num- 
her of samples but can in part be attributed to the time of year at which the ani- 
mal is slaughtered and to the animal's state of nutrition. The theory ha3 been 
advanced that when an animal ia fed high carbohydrate diets, increased amounts of 
thiamin are required to aid' in the metabolism of that carbohydrate, thus necessi- 
tating greater mobilization of the vitamin into the tissues. This may then ac- 
count for the high vitamin B-j_ found in the muscle of pigs which consume large 
amounts of carbohydrate. 



There are a great number of publications which list the thiamin content of 
foodstuffs as determined hy a variety of methods in different laboratories. In 
the following paragraphs some of the literature will be referred to. Many of 
the publications to be cited here will be those that have reported the use of 
crystalline thiamin as the basis for the estimation or which have given an approxi- 
mate value on the basis of a reliable conversion value. It should be cautioned 
that any conversion value furnishes only a good approximation and cannot be con- 
sidered as final in any case. Such an appraisal of the literature must of neces- 
sity eliminate many of the early assays which were able to furnish only a rela- 
tive comparison of the foodstuffs. 

Pierson (59) has reported the vitamin B content of several lamb tissues. 
Lamb heart and lamb liver contained 3.0 micrograms per gram of fresh tissue. The 
results of the assays reported by us show that lamb liver contains 4.1 micrograms • 
of vitamin B-j_. Taking into consideration the variation in samples, the values ' 
2.6 and 3.3 which we obtained compare very favorably with Pierson' s value of 1.5. 

The work of Kemmerer and Steenbock (^3) furnished several values for the 
vitamin Bj_ content of pork muscle, beef muscle, and chicken tissues. Their growth 
values were converted by Daniel and Munsell after using the necessary estimations. 
PZemmerer and Steenbock found beef muscle to contain 2.25 micrograms per gram and 
pork muscle 7-3 times that of beef. The dark muscle of the chicken contained 9.0 
micrograms per gram of fresh tissue, while the light muscle contained. approximate- 
ly k.k micrograms. Chicken liver contained 12.0 micrograms of vitamin B-, . The 
values for chicken muscle and pork muscle obtained by Kemmerer and Steenbock check 
with the values found by us and which are given in Table VII. 

The thiamin content of some animal tissues has been summarized by Williams 
and Spies (98). The parts per million of the tissues are listed as determined by 
the rat bradycardia method, the pigeon protective, the chick teat, and several rat 
growth methods. Although this table has some usefulness, it has the disadvantage 
that a so-called "preferred value" is given which creates an unfavorable impres- 
sion since many of the values are much too low. No weight is given the reliability 
of the methods quoted, and it appears from our data that pork muscle, an outstand- 
ing source of thiamin, is given a value that is exceedingly low. 






86 

Elvehjem, Sherman, and Arnold (21) have given some values for the thiamin 
content of tissues as determined by the chick growth method. These workers found 
that beef kidney and pork kidney contained 12.0 micrograms per gram of dried 
tissue. This was lower than the pork muscle which gave 19.8 micrograms per gram. 
Beef and pork heart. were next in potency with 7-8 and 7-5 micrograms per gram, 
while pork liver and prok lung were next in antineuritic value at 3-9 micrograms 
per gram. Beef and sheep muscle showed the least potency as tested by this 
method. These workers (2) also studied the effect of commercial canning processes 
on the vitamin Bj content of animal tissues and concluded that approximately 70 
to 80 per cent of the vitamin was destroyed by the two hours processing at a tem- 
perature of 2^0°F. Several samples of a canned produced containing high amounts 
of pork loin were assayed for the thiamin content using the ration of Waisman et 
al (88) previously described, and it was found that the destruction of the thiamin 
was considerably less than that reported by Arnold and Elvehjem. The sample which 
was heated at 225°F. for 50 minutes showed a loss of 30 per cent from the untreat- 
ed meat (87). Arnold and Elvehjem (3) investigated the effect of storing meat 
canned under vacuum on the stability of vitamin B-^. It was found that less than 
20 per cent of the vitamin was lost after a storage period of nearly two years. 
Beef spleen showed the least loss during the storage of the vacuum packed samples. 

The studies of Kellogg (k2) indicated that lamb tissues contain from 1 to 3 
micrograms of thiamin per gram of tissue. Christensen, Latzke and Hopper (17) 
have reported on the influence of cooking and, canning on the vitamin content of 
beef and pork. Using the conversion figure that 2 Sherman-Chase units are the 
equivalent of 1 international unit, dried lean pork contained 36 micrograms per 
gram or 10.6 micrograms per gram of fresh tissue , The dried beef contained 3-6 
micrograms per gram or 0.9 micrograms per gram of fresh beef muscle. After cook- 
ing, it wa3 found that 12 per cent of the vitamin was destroyed in the pork and 
20 per cent in the beef. When the tissues were heated in a steam autoclave, the 
destruction was greater, amounting to 21 per cent In the pork and nearly 100 per 
cent in the beef. 

Baker and Wright (7, 8) determined the amount of vitamin B in many foods and 
found that meat preparations were a good source of the vitamin. These workers 
used the bradycardia method in which they standardized the ability of vitamin Bi 
to restore the normal heart rate of rata suffering from incipient polyneuritis. 
By feeding their supplement, they were able to determine the quantity of the vita- 
min in that supplement. Some of the values they obtained expressed in interna- 
tional units per gram are as follows: lean raw beef, 0.5; cooked ox liver, 1.5 J 
cooked veal, 0.5; lean raw mutton, 0.6; roast lean lamb, 0.5; braised sheep tongue, 
0.2; raw sheep kidney, 1.9; roast leg of chicken, 0.U; roast lean pork, 3-2; 
boiled lean ham, 2.2; raw pig brain, 0.6; raw pig kidney, J>.h. It will be seen 
seen that these values are in fair agreement with those of Table VII although they 
are a microgram or so lower. 

A recent publication from the Bureau of Home Economics, U. S. Department of 
Agriculture, has expressed the vitamin B^ content of foods in terms of crystalline 
thiamin (lk) . Lean beef muscle contained 1.14 micrograms per gram, dark chicken 
1.1, and light chicken 0.8. These values agree very well with those that we have 
obtained by a similar method. The Department of Agriculture workers found 1^.3 
micrograms for smoked ham, 2.k and 2.6 for lamb chops, and 2.67 for beef liver. 
They found that the lean portion of pork chop contained nearly Ik micrograms of 
thiamin per gram. 

In a recent publication by Pyke (65) the vitamin B-j_ content of vertebrate 
muscle is given for a large number of species. The vitamin B^ was determined by 
the thiochrome procedure and gives results which further point out the high con- 
tent of this vitamin in the muscle of pork. This worker found that pig muscle 



87 



varied from 170 to 510 international units per gram. Goose muscle shoved a range 
of values from 0.13 to O.67 international units, -while rabbit muscle was only 
slightly lower. The vitamin B-, content of kid muscle showed a somewhat narrower 
range, being 0.23 to O.63 international units. The figure given for sheep was 
0.3^, ox 0.23, and fowl .0.19 to 0.57 international units per gram of tissue. 



The human daily requirement for thiamin has been usually placed at between 1 
and 2 milligrams per day for the normal adult. Cowgill (l8, 19 ) has suggested 
about 300 international units as the figure for the normal 70 kilogram adult. 
Several workers have attempted to base the thiamin requirement on the caloric in- 
take, and others have attempted to obtain the daily requirement from saturation 
and excretion studies. Although there does not seem to be any unanimity on the 
subject, the Committee on Foods and Nutrition of the National Research Council has 
offered a tentative figure which is approximately 2 milligrams. 

In order to arrive at some estimate of the ability of meat to furnish the 
daily human requirement of thiamin, it is necessary to consider several factors 
which may influence the figure which is obtained. There are several approaches 
to the calculation of the actual amount of thiamin secured in our foods. One of 
these ia based en the caloric intake. Stiebling and Ward (78) have indicated 
that the typical diet contains about 7 per cent meat. If it is assumed that the 
average daily food consumption furnished 2500 calories, and if each gram of food 
is able to yield h calories of energy, then approximately 625 grams of food would 
supply this daily energy requirement. If 7 per cent of our daily food is meat 
on the weight basis, then approximately 45 grams of meat would be consumed. Fifty 
grams of fried beef round will supply nearly 125 micrograms of thiamin. This 
means that a quarter pound of round steak will furnish between 250 and 300 micro- 
grams or 80 to 100 international units of thiamin. Roast beef will supply some- 
what less than this amount. If an average size fried pork chop yielding 100 grams 
of meat is consumed, it will furnish 9^0 micrograms of thiamin or over 300 inter- 
national units of vitamin Bi . 



Perhaps the most reliable and most recent work on the question of how much 
vitamins are furnished in the food of the general population is that of Stiebling 
and Phipard (77). These workers have related the expenditure level for food with 
the nutritive requirements. Their data present evidence that as more money is 
spent for food, more vitamin B-| is taken in the diet. From the figures presented, 
it appears that "not until weekly food expenditures reached $2.50 to $3.12 a per- 
son did most groups of families obtain diets averaging more than 500 international 
units." A more detailed perusal of this circular reveals that further data is 
presented on the average distribution of expenditures among specified groups of 
foods, and the proportion of nutritive content contributed by each group of foods. 
For example, in the Ea3t North Central families which spent $1.88 to $2. '49 weekly 
per capita for food, only 2^.2 per cent of the money was spent for lean meat, 
poultry and fish, yet more than 30 per cent of the vitamin B]_ was contributed by 
this class of foods. In other words, meat is able to furnish a good share of the 
daily human requirements of thiamin, and as more money is spent for meat, more 
of the vitamin will be supplied. 



89 



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97. Williams, R.D., Mason, H.L., Wilder, P.M. and Smith, B.F. Observations on 
Induced Thiamin Deficiency in Man. Arch. Int. Med., 66, 785 (19U0). 

98. Williams, R.P. and Spies, T.D. Vitamin Bj and Its Use in Medicine. 1938. 
MacMillan Company. 

99. Williams, R.R., Waterman, R.E. and Keresztesy, J.C. Larger Yields of 
Crystalline Antineuritic Vitamin. J. Am. Chem. Soc, 36, 1137 (193^)- 

100. Wright, H. Changes in the Neuronal Centers in Beriheric Neuritis. 
British Med. J., JL, l6l0 (1901). 



\ 



CHAPTER X 

RIBOFLAVIN 

Page 
Chemistry of Riboflavin 95 

Physiology and Pathology of Riboflavin 96 

Riboflavin Therapeut i c s 99 

Methods of Assay _ __ _ 99 

Microbiological Assay for Riboflavin _ 100 

Results _ 102 

The Influence of Cooking on the Riboflavin 

Content of Meat 105 

Discussion of the Results - 107 

The Ability of Meat to Supply the Riboflavin 

Requirement 107 

Literature Cited 113 



CHAPTER X 
RIBOFLAVIN 



Chemistry of Riboflavin 



The work of Earrer and co-workers (50,53), Kuhn and co-workers (59, 60), 
Koschara (23,57) and von Euler and co-workers (32) contributed much to the isola- 
tion of the yellow, fluorescent pigment that was found to occur in many animal 
and plant products, and which we now know as riboflavin. The pigment was obtained 
from liver, kidney, egg white, milk, urine, a variety of grasses and flowers, 
malt and the retinas from fish eyes. Extraction procedures made use of alcohol, 
alcohol- water mixtures and a variety of steps involving adsorption and elution 
from suitable adsorbent materials. Further purification was obtained by making 
the heavy metal ^salts, followed by recrystallization of the silver or thallium 
salt from water, dilute acetic acid or alcohol solutions. 

The researches of Warburg'and Christian on the pigment component of their . 
yellow enzyme (112) furnished a clue as to the possible identity of the colored 
pigments obtained from liver, kidne'y, milk and eggs. Warburg and Christian (113, 
llU) had already given many of the properties of their pigment-, and it. appeared 
from simultaneous experiments with rats in several laboratories that the loss of 
vitamin activity paralleled the loss of a hydroxyl type compound from Warburg and 
Christian's yellow enzyme when the latter was irradiated in alkaline solution. 
Intensive studies by Kuhn, Rudy and Wagner -Jaur egg (67) and Kuhn and Rudy (61+, 65) 
on the products of alkaline hydrolysis of the vitamin resulted in the finding that 
a sugar chain split off as a result of photolysis of riboflavin. 

The synthesis of riboflavin was accomplished by Kuhn and his co-workers (63) 
and by Karrer and his co-workers (51). One of the difficulties in the final 
synthesis was the preparation of adequate amounts of d-ribose. The formula for 
riboflavin is given below: 



H 



CH 5 -( 



CH, 



/ 



CHo 



N 



H H H 

■C — C C — CHpOH 



H H R 



/ 



N- 



•C=0- 



N-H 



d-Ribo flavin 

The relation of chemical structure to biological activity has been investi- 
gated. It ha3 been found that at least one of the methyl groups in position 6 or 
7 is essential for vitamin activity. If the methyl groups are shifted from their 
true positions to either 5 or 8 then the vitamin activity is also destroyed. The 



-95 ■■ 



96 

shifting of the methyl groups also nullifies the coenzyme activity of the mole- 
cule. Kuhn has shown that a slight change in the side chain will reduce the 
vitamin activity, and only d-ribose or 1-arabinose have been found compatible 
with physiological action. The 1-arabinose compound was also synthesized but was 
found to have only one-third the activity of the d-riboflavin (68,71). 

Several properties of riboflavin were described before the actual structure 
of the substance was known, but they have since been confirmed on the crystalline 
product. The characteristic yellow green fluorescence was found to be at a maxi- 
mum between a pH of 6.0 and 7.0 and any great variation from this range resulted 
in the fading of the color. It was early noted that the optical activity of ribo- 
flavin was at a maximum levo rotation, [>] 2 ° - -nU°, when the concentration of 
sodium hydroxide was tenth normal (52,66). Warburg and his co-workers recognized 
that flavin was easily oxidized and reduced, but it remained for Kuhn and his co- 
workers (62), Stern (100) and Kuhn and Wagner -Jaur egg (68) and Michaelis, Schu- 
bert and Smythe (76) to perform accurate measurements of the oxidation reduction 
potentials. Riboflavin is very soluble in aqueous solution owing to the presence 
of the d-ribityl group. It is insoluble in organic fat solvents until the ribose 
group is removed. Riboflavin is stable in strong mineral acids and toward oxidiz- 
ing agents, but 13 readily destroyed by alkali or irradiation. 

Physiology and Pathology of Riboflavin 

A historical sketch of the physiology of riboflavin must include the early 
investigations of Goldberger, who first observed that rats maintained on the 
"pellagra producing" diet lost a good share of their hair, ceased to grow, and 
showed an ophthalmia and in later stages cataracts developed. Goldberger con- 
cluded that pellagra was due to a bad nutritional state and that the rat could be 
used for experimental studies. The condition noted in the rats was believed to 
be due to a vitamin deficiency and was named vitamin B2 or G. It was also called 
the "pellagra preventative factor" or the P-P factor. It soon became apparent 
to certain workers in the field that the rat experiments did not completely ex- 
plain the variety of symptoms that were observed. Hogan and Hunter (U3) finally 
suggested that more than one factor was concerned with the so-called "vitamin B2" 
deficiency. Elvehjem and Koehn (26) also demonstrated that the yellow pigment 
"flavin" did not cure the condition in dogs known as black tongue, which was be- 
lieved to be identical with the condition in humans known as pellagra. Gyorgy 
also differentiated between a riboflavin deficiency and so-called "rat pellagra". 
We now know that the early symptoms described by Goldberger were a combination of 
several nutritional deficiencies. 

A condition of cataract in riboflavin deficient rats has been described by 
Day (21). This condition was usually preceded by a conjunctivitis and keratitis. 
The cataracts in the rats occurred to the extent cf nearly 100 per cent. These 
workers ( 17, 3.8, 19,20) have claimed that the type of cataract produced in mice, 
chickens and monkeys can be prevented by riboflavin. This work has been challenger: 
however, by Bourne and Pyke (10) in which they obtained only one-third the catar- 
acts seen by Day and associates. Gyorgy (37) snd Richardson and Hogan (91 ) also 
found that cataracts in these animals were rare. There are a number of papers 
which deai with the cataracts in rats produced by a high lactose diet (80,8l), 
but there is no clear reconciliation at present between the work of the various 
authors cited here. 

It becomes apparent from a review of nutritional ' research that no one animal 
can be relied upon to present a complete picture of the physiology and function 
of a. particular vitamin. The realization that experiments with the different 
laboratory species gave a broader picture led many investigators to repeat their 



97 

original rat experiments with dogs, chicks, monkeys and pigs. The variety of 
symptoms observed in these animals on a particular deficiency attested to the ad- 
visability of such repetition. In growing chicks, for example, the birds on a 
riboflavin deficient diet became weak and emaciated and developed diarrhea but 
no dermatitis (73). Bethke, Record, and Wilder (6) observed a characteristic leg 
disorder in chicks on their riboflavin deficient diet. This leg condition has 
now been termed "curled toes." Turkeys and laying hens have also been shown to 
require riboflavin by Bethke, Record and Wilder (7), and Norris, Wilgus, Ringrose, 
Heiman, and Heuser (83). 

The symptoms of riboflavin deficiency in the dog have been observed by a 
number of workers.. Street and Cowgill (103) produced an acute riboflavin defi- 
ciency in the dog by feeding a diet containing sucrose, purified casein hydro- 
genated fat, bone ash, and salts, supplemented with a vitamin B-, concentrate, and 
vitamins A and D. While the ration was deficient in several factors, Street and 
Cowgill were able to obtain dramatic cures by injecting or feeding the crystalline 
vitamin. Axelrod, Lipton and Elvehjem (2), using a purified diet similar to that 
of Street and Cowgill but containing additional factors, demonstrated that dogs 
fail to grow and show intestinal hemorrhages and ulcers. Sebrell and Onstott (96) 
found in their experiments a syndrome of bradycardia, arrythmia, collapse and coma 
together with a yellow mottling of the liver and degenerative changes in the cen- 
tral nervous system. Fraser, Topping and Isbell (35) have also studied the ribo- 
flavin deficiency in dogs that were maintained on a modified Goldberger diet. 

The riboflavin content of tumor tissue has received some attention by several 
groups of workers, G-yorgy, Kuhn and Wagner -Jaur egg (38), von Euler and Adler (32), 
and Elliot, Benoy and Baker (25). The first two groups of workers analyzed tumor 
tissue for its riboflavin content and have concluded t.'.at Roux sarcoma of chicks, 
contains a low concentration of the vitamin. Elliott and associates conclude from 
this that the failure of abnormal tissue to metabolize lactate or malate is per- 
haps due to the lack of enzymes or coenzymes which contain riboflavin. 

Kuhn and co-workers showed (6l) that the riboflavin content of the organs of 
rats could not be increased greatly, no matter what amount of riboflavin was giver 
in the diet. It was also shown that the body will attempt to conserve the ribo- 
flavin in the liver, and animals at death still retain about one-third of the 
normal amount of vitamin in this organ. Kuhn. 's experiments also showed that after 
severely depleted animals were given riboflavin, the growth began at once, while 
the reserves in the organs were only gradually restored. Emmerie (28,29) has per- 
formed much of the work on the excretion of riboflavin and its variation with the 
consumption of the vitamin. It was demonstrated that with an increased intake 
there was an accompanying increased excretion. 

The effect of riboflavin low diets upon the nerve structure of rats has been 
studied by Engel and Phillips (31). The spinal cord and peripheral nerves of the 
deficient rats were apparently normal. This is in contrast to previous work on 
chicks by these investigators (87) in which they found severe degeneration in the 
myelin sheaths of the spinal cords and the peripheral nerves. 



The influence of diet on the riboflavin content of milk has been investigated 
by Hunt and Krauss (^5), Donelson and Macy (22), and Kuhn, Wagner -Jaur egg and 
Kalschmitt (69). These investigations, however, were carried out without due 
appreciation of the influence of the rumen synthesis upon the constituents in milk. 
The work of McElroy and Goss (77) and of Wegner, Booth, Elvehjem and Hart (115) 
gives good evidence to support the theory that bacterial synthesis of riboflavin 
in the rumen is of prime importance. The ruminant3 are able to synthesize ribo- 
flavin in the rumen to the extent of a fourfold increase over that in the feed. 



98 

Vegner et al have found approximately 5 micrograms of riboflavin per gram of 
feed, and an analysis of the rumen contents has shown about 20 micrograms per 
gram, all figures "based upon dry weight. In a calf maintained on a "synthetic" 
diet in which there was no riboflavin, the rumen contents still showed a level 
of 20 to 25 micrograms per dry gram of rumen contents. One consideration here 
is the effect of the ration constituents on the intestinal flora. A recent paper 
by Johnson, Maynard and Loosli (^7) gives evidence for the fact that the riboflavin 
content of the milk cannot be changed more than 25 per cent even when the ration 
is designed to be very low in the vitamin. These workers also found that when a 
molasses -yeast by product, high in riboflavin, was fed, there was only a temporary 
increase in the riboflavin content of the milk. The conclusion from their data 
Is that there exists an inverse relation between the milk yield and the riboflavin 
concentration of the milk. 

Riboflavin seems to have a definite effect on the absorption mechanisms of 
the body, according to work done by Lazst and Verzar (72) on iodoacetic acid poi- 
soning and its relation to the formation of riboflavin phosphate by adrenal cor- 
tical extract. These workers found that growth in rats was retarded by feeding 
a small amount of iodoacetic acid to a complete diet. If the animals were given 
riboflavin-phosphoric acid growth was restored, but riboflavin itself wa3 ineffec- 
tive. Adrenal cortex extract also seemed to overcome the toxic effect of the io- 
doacetic acid 3ince growth was resumed when thi3 substance was given. The con- 
clusions drawn from these experiments by the authors were that riboflavin must be 
phosphorylated in order to function normally and that adrenal cortex extract is 
undoubtedly essential for the phosphorylation. 

Some members of the vitamin B complex have been said to function as precur- 
sors of their respective coenzymes. That is, thiamin must be supplied in the diet 
to form cocarboxylase, and riboflavin is necessary to form d-amino acid oxidase, 
xanthine oxidase, or several of the other riboflavin containing enzymes. It is 
believed that the pathic symptoms in the particular vitamin deficiencies are due 
in part to a disturbed metabolic state resulting from a deficiency of the neces- 
sary enzyme or coenzyme. This belief is strengthened by the observations of Ochoa 
and Peters (Qh) who found a decreased cocarboxylase content in tissues of animals 
suffering from thiamin deficiency. Axelrod, Madden, and Elvehjem (l), Vilter, 
Vilter and Spies (110) and Eohn, Klein, and Dann (56) observed a lowering of the 
pyridine dinucleotide content of some deficient tissues of animals on a nicotinic 
acid deficiency. More of these particular studies will be given in the chapter 
devoted to nicotinic acid. 

Ochoa and Rossiter (85) have reported a decrease in the flavin-adenine-di- 
nucleotide content of boiled extracts of liver and heart tissue from rat3 fed a 
riboflavin deficient diet. No such decrease could be detected in similar extracts 
of brain and kidney tissue. Several enzyme systems are related to riboflavin as 
already hinted above, and these have been summarized by Axelrod, Sober and Elveh- 
jem (3) in their paper on the d-amino acid oxidase content of rat tissue In ribo- 
flavin deficiency. They found a definite decrease in the d-aminc acid oxidase con- 
tent of livers from animals suffering from a riboflavin deficiency. 

One function of the enzymes systems in the body is to help bring about oxida- 
tions and reductions which are necessary for normal metabolism. Since riboflavin 
is concerned with several enzymes and coenzymes it is obvious that this vitamin 
may function through these substances, warburg and Christian (112, 11^-) first 
pointed out the significance of the yellow enzyme as part of the system which oxi- 
dizes hexose-phosphorie acid to phosphohexonic acid. Theorell (108, 109), purified 
the enzyme and identified the prosthetic group of Warburg and Christian's enzyme 
as riboflavin phosphate. Hogan (h~5) in reviewing the physiology of riboflavin 
presumes that "the necessity for including riboflavin in the diet is that it is 



99 

an essential constituent of the yellow oxidation enzyme that cannot he synthesized 
hy the animal cell. It is probable, then that the vitamin activity of riboflavin 
is due to the fact that it forms an est-^r with phosphoric acid, and this ester 
combines with protein to form the yellow oxidation enzyme." In addition to ribo- 
flavin phosphate, very recent work has indicated that riboflavin is a component of 
the coenzyme riboflavin adenine dinucleotide (36). 

Riboflavin Therapeutics 



The increasing importance of riboflavin in the human dietary is reflected 
in several reports on human ariboflavinosis. Sebrell and Butler (95) reported 
the experimental production of lesions in the angles of the mouth on the lips 
and near the nose and eyes. Oden, Oden and Sebrell (86) have further reported 
human cases of riboflavin deficiency in rural sections of Georgia. Sydenstricker, 
Geeslin, Templeton and Weaver have also reported on the riboflavin deficiency in 
human subjects (106). The ocular manifestations of riboflavin deficiency have 
been described by Kruse, Sydenstricker, Sebrell and Cleckley (58). 

Although it has been conclusively shown (27) that nicotinic acid is the 
antipellagra vitamin, the role of riboflavin in complex deficiency states associ- 
ated with pellagra is an important one. Spies, Vilter and Ashe (99) have con- 
cluded that a riboflavin deficiency will be cured by the administration of the 
crystalline vitamin and that a pellagrin in relapse who will not respond to nico- 
tinic acid will show improvement with the administration of riboflavin. Syden- 
stricker, .Geeslin and Weaver (107) showed that patients who were under insulin 
therapy and who were being given the restricted diets of diabetics developed the 
typical symptoms of cheilitis, and conjunctivitis of riboflavin deficiency. These 
patients responded to riboflavin treatment. When the patient was given small 
dosages of insulin, the signs of riboflavin deficiency disappeared, but when the 
insulin wa3 increased the cheilitis was more prominent. Riboflavin cured the 
cheilitis, but again there was a relapse. There is some relationship between the 
utilization of carbohydrate and the coenzymes which are necessary for its meta- 
bolism. It may be that the injections of insulin rapidly depleted the body stores 
of the vitamin as a result of the increased carbohydrate metabolsim. Jolliffe, 
Fein and Rosenblum (-+8) described several manifestations of riboflavin deficiency 
as seen in alcoholics, pellagrins, scorbutics and polyneuritics. Fissures and 
macerations at the corner of the mouth were usually cured by 10 milligram daily 
feedings . 

The daily need for riboflavin is as yet not definitely known. Rose (92) 
suggested that approximately 60 micrograms per 100 calories of food, for the child, 
and about 1/3 of that for the adult. This means that for an average adult consum- 
ing 2500 calories, the intake of riboflavin should be in the neighborhood of 500 
micrograms per day. The requirement for children up to ten years would then be 
about 1500 micrograms. 

These figures are too low as shown by more recent work. Emmerie (28,29) set 
the daily suggested intake of riboflavin at 2 to 3 milligrams on the basis of 
urinary excretion studies. Stiebling and Phipard suggested 1.8 milligrams as the 
desirable human adult allowance. The Research Council Committee on Food and 
Nutrition has tentatively set the optimum human daily need for riboflavin at 2 
to 3 milligrams. 

Methods of as 3 ay 

When it became necessary to make quantitative measurements of the vitamin 
in natural materials, only the property of fluorescence was resorted to as a pos- 
sible method, yon Euler and Adler (33) and Cohen ( 13,1*0 measured the intensity 



100 

of the fluorescence. Warburg and Christian (113) and Kuhn et al (69) uaed the 
colorimeter and estimated the lumiflavin content of a chloroform extract from the 
aqueous phase after conversion of the lactoflavin into lumiflavin "by exposure to 
light in alkaline solution. A fluorometric method has "been described by Supplee, 
Ansbacher, Flanigan and Hanford (105) in which they use "black light", transmit- 
ting between 3100 and 4l00 A" . A simple fluorescent method has been described 
by Weisberg and Levin (ll6) in which the fluorescence of riboflavin is compared to 
that of sodium fluorescein under ultra violet light. Supplee, Bender and Jensen 
(104) have presented their study of the correlation between the "black light" 
method and a rat growth method. 

Ellinger (23) has critically reviewed the available physicochemical proce- 
dures and he concluded that the best of these methods la inferior to the biologi- 
cal methods. One outstanding difficulty is the means by which the vitamin is ex- 
tracted and thus made available for the determination. The presence of interfer- 
ing materials that possess fluorescent properties would of course nullify the 
method. Perhaps the earliest biological method available for the determination 
of "vitamin G or B2" was that of Bourquin and Sherman (ll). This method involved 
the use of a diet low in riboflavin and by suitable growth upon a particular 
supplement, the "units" of riboflavin could be calculated. Although this ration 
had served its purpose in determining riboflavin, later work demonstrated that 
the diet was low in several factors, which perhaps later accounted for the dis- 
crepancies in ascribing to the Sherman -Bo urcuin unit a definite value in terms 
of micrograms of crystalline riboflavin (U, 39, 7*0. Booher, Blodgett and Page (9) 
and Bessey (3) have shown that the Sherman -Bourquin diet is suitable for quantita- 
tive determinations of riboflavin in food3. Bethke, Record and Wilder (6) describ- 
ed a characteristic leg disorder in chicks on a riboflavin deficient diet and 
asserted that when 40 micrograms was given to each chick e^ery other day, the leg 
disorder was prevented. Jukes also described a biological assay of riboflavin 
with chicks (*t-9). Norris, Wilgus, Ringrcse, Heiman and Heuser (83) have also 
based their determination of the riboflavin content of liver on the growth of 
chicks. Wagner, Axelrod, Lipton and Elvehjem (ill) have proposed a rat method 
for the determination of riboflavin which contains adequate quantities of the 
known vitamins together with riboflavin free concentrates of the unidentified 
factors . With suboptimal amounts of riboflavin, the growth rate is proportionate 
to the amount of riboflavin fed, but with adequate amounts, the growth is normal. 

Microbiological Assay for Riboflavin 

There became available early in 1939, a microbiological method for the de- 
termination of riboflavin. The method appeared to be most worthwhile since it was 
rapid, specific and accurate. This microbiological assay was developed by Snell 
and Strong (98), and depends upon the growth of a specific strain of the organism 
Lactobacillus casei € (Lactobacillus helveticus) on a media which is complete in 
all the nutritive factors required by this organism for optimum growth except 
riboflavin. The completeness of the media can be illustrated by the maximum 
growth which is obtained either with crystalline riboflavin or with a yea3t or 
liver extract which will furnish an equal amount of riboflavin. This method ap- 
peared to be trustworthy on the basis of preliminary assays that were carried out 
with the different meat products available from the thiamin assays. The prelimin- 
ary assays on liver, muscular tissue and bra.in gave the indication that reproduc- 
ible results could be expected with a constant means of extraction. The details 
of the method can be found in the original paper of Snell and Strong, but the 
salient points of the microbiological procedure will be given here. 

The media consists of a photo lyzed, NaOH treated peptone, 0.5 per cent glu- 
cose, 1 per cent; sodium acetate, 0.6 per cent; cystine, 0.01 per cent; inorganic 
salt mixture; and riboflavin-free yeast supplement equivalent to 0.1 per cent 



101 

yeast extract. The various solutions can "be prepared separately in "brown "bottles 
and kept under toluene until they are used. A pure standard solution of ribo- 
flavin is also kept under toluene. All the constituents of the "basal media were 
kept in the icebox, as a further precaution against spoilage. 

Approximately 200 tubes were used in a particular series allowing the assay 
of at least 30 samples together with suitable standards. The tissues wl}ich were 
assayed for riboflavin were in many cases the same that we used for the thiamin 
assays. Mare samples were assayed for riboflavin than were assayed for thiamin 
largely due to the convenience of the method. As a result of preliminary investi- 
gations it was found that liver and kidney of calf, lamb, sheep and beef were de- 
finitely higher in riboflavin content than the muscle of other tissues of these 
species. Accordingly enough of each dried tissue was used at those levels which 
would supply 0.1 microgram per cc . of final extract. 

In order to obtain complete extraction of the riboflavin from the meat, the 
accurately weighed dried tissue was -placed in a test tube and homogenized in 10 
cc. of warm water. The homogenizing apparatus was that of Potter and Elveh.jem 
(89). The uniform mixture was then quantitatively transferred to a 50 °c. centri- 
fuge tube and diluted to 35 cc. of water. The tubes were plugged with cotton and 
autoclaved at 15 pounds pressure at I20°C. for 15 to 25 minutes. When the tubes 
were cool they were centrifuged and the supernatant liquid was poured into a 100 
cc. volumetric flask. The residue was suspended in about 20 cc. of water and cen- 
trifuged again. The washing was often repeated a second time. The washings were 
added to the original extract in the volumetric flasks. All extracts were finally 
diluted to 100 cc. and various aliquot3 of this solution were added to the basal 
media in the test tubes. The extract in the 100 cc . volumetric was transferred 
to appropriate bottles and preserved under chloroform. A number of trial runs 
convinced us that this and similar preservatives had no influence on the results 
of the method as long as care was exercised to prevent the addition of larger 
amounts of chloroform to the test tube. Later trials showed that the autoclaved 
and homogenized samples could be used directly for assay in the form of suspen- 
sion. 



When these extracts or suspensions were assayed, they were placed in the 
tubes at levels of 0.5 cc, 1.0, and 1.5 cc. All the assays were run in duplicate 
together with a set of standard tubes with graded amounts of crystalline ribo- 
flavin. The tubes were plugged, autoclaved for 15 minutes, and after cooling were 
aseptically inoculated with a normal saline suspension of a freshly cultured 
Lactobacillus casei £ (Lactobacillus helveticus) . All this work was done away 
from bright light so as to avoid possible destruction of riboflavin in the aqueous 
solution. The tubes were incubated at 37° C. in the incubator room for 3 days. 
At the end of this period, the acid produced by the microorganisms was titrated 
with N/lO ITaOH. By means of a reference curve plotted from the titration values 
of the standard tubes containing the crystalline riboflavin, the riboflavin con- 
tent of the unknown samples were determined. Within a given range, the amount of 
acid produced by the organism i3 directly proportional to the concentration of 
riboflavin in the media. 

Snell and Strong (98) reported a number of assays on natural materials both 
by the bacteriological and rat growth methods and concluded that both tests gave 
the same values for riboflavin. Similar parallel assays on some of our meats and 
meat products, fully confirming the previous results were secured by the rat growth 
and by the microbiological method. It is unnecessary to give this data, but let 
it suffice to say that the values are in good aggrement. A similar comparative 
study have also been fully confirmed by Wagner, Axelrod, Lipton and Elvehjem (ill). 



102 

These workers compared the riboflavin values for several liver preparations and 
cereal grasses with those obtained by their rat growth method and by the micro- 
biological assay. A rather complete comparison of bioassay, microbiological and 
fluorometric methods has been reviewed by Emmett, Bird, Brown, Peacock, and Vanden- 
belt (80). They come to the conclusion that reproducible results can be obtained 
by the biological rat growth, visual fluorescence, photoelectric fluorescence, 
and microbiological growth. The four methods give similar results except that 
the greatest differences were observed in the low potency samples. 

Before initiating the large number of assays on the meat products it was 
necessary to know whether there was any destruction of riboflavin during our dry- 
ing processes. A sample of pork liver (sample 122) was analyzed in both the 
fresh and the dried condition in simultaneous assays at equivalent levels. The 
fresh sample was homogenized and prepared for assay shortly after the meat arrived 
from the packer, while the same sample was also assayed as soon as it was dried 
according to our drying procedure described above. Moisture determinations weru 
made on the fresh tissue to enable comparisons with the dried sample. The fresh 
tissue contained 80 micrograms of riboflavin per gram on the dry weight basis 
which agrees with the 85 micrograms per gram in the dried sample. Beef spleen was 
also assayed fresh and dried. The values were 19. o and 21.0 respectively. A 
sample of boiled ham was treated in a similar manner and again the results indi- 
cated no destruction during the drying process. The fresh boiled ham 'gave 7.8 
micrograms per gram as compared to- 8.0 micrograms for the gram of dried boiled 
ham when calculated to an equal quantity of dried meat. Similar results were also 
obtained with tenderized ham and pork ham. 

Results 

The microbiological assays of 90 samples of meat are given in Table IX; Most 
of the values are the average of several assays. In many cases a new extract was 
prepared before each assay. However, .this procedure was found to be unnecessary 
since similar values were obtained in the various extracts of the same sample. 
It is apparent that livers and kidneys of all the animals studied contain high 
amounts of riboflavin. Samples of beef liver averaged about 100 micrograms of 
riboflavin per gram of dry liver or 30 micrograms fresh liver. Samples of veal 
liver, pork liver and lamb liver gave values in the same range. The liver samples 
of the various species contain a fairly constant amount of liver. Beef kidney 
and pork kidney averaged 95 micrograms per gram of dried tissue or 20 micrograms 
per gram of fresh kidney. Other tissues were not as rich in riboflavin as liver 
and kidney. Heart was next in potency, followed by beef pancreas, beef spleen, 
beef brain and lung. 

The muscular tissues contained less riboflavin than organ tissue. Beef 
muscle contained approximately one-tenth that found in beef liver. Veal muscle 
contained the same amount of riboflavin as did beef muscle. Pork ham and pork 
loin also contained about one-tenth that found in liver or kidney. The commercial 
products such as smoked ham, boiled ham, tender made ham, were in the same range 
of riboflavin values as the other meats. 



103 







TABLE IX 








Riboflavin Content 


of Meats and 


Meat Products 






Sample 




Micrograms/ 


Micrograms/ 


Meat 


number 


Process 


gm. fresh 


gm. dry 


Beef "brain 


57 


Dried 


2.5 


12.0 


Beef "brain 


77 


1! 


- 


11.0 


Beef train 


132 


It 


2.7 


12.5 


Beef heart 


73 


It 


7.6 


36.0 


Beef heart 


133 


It 


10.0 


52.0 


Beef kidney 


h9 


tt 


21.1 


100.0 


Beef kidney- 


81 


II 


22.8 


100.0 


Beef kidney 


8^ 


tt 


18.7 


90.0 


Beef kidney 


126 


II 


18. k 


92.0 


Beef liver 


98 


tt 


- 


100.0 


Beef liver 


lie 


It 


37.0 


125.0 


Beef liver 


120 


II 


30.0 


100.0 


Beef liver 


131 


II 


21.0 


72.0 


Beef lung 


78 


tl 


- 


15.0 


Beef lung 


138 


It 


h.9 


2U.0 


Been pancreas 


6k 


tt 


5.5 


17.0 


Beef pancreaa 


19 


It 


- 


19.0 


3eef pancreas 


113 


tt 


5.0 


19.0 


Beef round 


ho 


tl 


3.5 


11.0 


Beef round 


105 


tt 


1.9 


7.0 


Beef round 


128 


tl 


2.1+ 


10.0 


Beef 3pleen 


59 


II 


3.5 


15.0 


Beef spleen 


76 


II 


" 


13.0 


Beef spleen 


108 


II 


- 


16.0 


Beef spleen 


123 


Fresh 


h.l 


19.6 


Beef 3pleen 


123 


Dried 


5.8 


21.5 


Beef spleen 


130 


tt 


3.9 


18.1+ 


Beef tongue 


82 


tt 


2.2 


8.0 


Veal hindquarter 


56 


ti 


2.1+ . 


11.0 


Veal hindquarter 


103 ' 


it 


2.k 


9.0 


Veal hindquarter 


129 


it 


3.7 


15.0 


Teal hindquarter 


134 


n 


3.0 


13.6 


Veal liver 


70 


11 


26.5 


100.0 


Veal liver 


97 


11 


39.0 


135.0 



104 



TABLE IX (continued) 



Meat 



Lamb leg 
Lamb leg 
Lamb liver 
Pork ham 
Pork ham 
Pork ham 
Pork ham 
Pork ham 
Pork ham 
Boiled ham 
Boiled ham 
Boiled ham 
Smoked ham 
Smoked ham 
Smoked ham 
Canned ham 
Tender made ham 
Tender made ham 
Pork heart 
Pork kidney 
Pork kidney 
Pork kidney 
Pork liver 
Pork liver 
Pork liver 
Pork loin 
Pork loin 
Pork loin 
Pork loin 
Pork loin 
Pork loin 
Pork loin 
Poultry, dark 
Poultry, light 
Poultry, light 



Sample 
number 


Process 


Micrograms/ 
gm. fresh 


Micrograms 
gm. dry 




55 


Dried 


3.6 


13.0 




80 


t! 


2.8 


11.0 




96 


II 


26.6 


92.0 




32 


II 


2.7 


10.0 




95 


II 


2.0 


8.0 




124 


II 


3-37 


14.3 




114 


II 


2.2 


8.0 




118 


II 


2.2 


8.0 




139 


II 


3.1 


12 . 2 




101 


II 


2.6 


8.0 




115 


II 


2.8 


8.0 




115 


Fresh 


2.7 


7.8 




47 


Dried 


2.9 


9.0 




102 


ii 


2.3 


8.0 




116 


M 


2.4 


8.0 




72 


n 


2.2 


7.0 




117 


ii 


2.4 


7.0 




119 


M 


2.0 


6.0 




104 


ii 


1.1 


5.0 




62 


n 


20.0 


92.0 




83 


ti 


19.7 


90.0 




137 


tt 


19.0 


94.0 




86 


ii 


29.0 


90.0 




122 


Fresh 


26.9 


85.O 




122 


Dried 


25.4 


80.0 




36 


it 


2.0 


7.0 




74 


ii 


2.5 


9.0 




89 


M 


2.7 


8.0 




90 


" 


2.3 


8.0 




91 


it 


2.4 


8.0 




92 


it 


2.7 


9.0- 




125 


ii 


2.75 


10.0 




66 


n 


2.6 


10.0 




t>5 


ti 


0.8 


3.0 




68 


it 


0.7 


3.0 





105 



The influence of Cooking on the Riboflavin Content o f Meat 



Several of the cooked tissues which were assayed for thiamin were also tested 
for their riboflavin content by the microbiological method. Together with cooked 
samples that were assayed for thiamin, several additional samples were cooked 
according to the methods described previously in order to obtain a broader survey 
of the influence of cooking on the occurrence of riboflavin in meats. The results 
are conveniently summarized in Table X. It will be seen that the three samples 
of beef liver that were fried showed some variation in the loss of riboflavin. 
These variations may be explained in part by the differences in treatment such as 
temperature, time of cooking and perhaps the greater amount of leaching in one 
sample than in another. The samples of beef round that were fried also showed the 
same variation, since one sample showed practically no loss from the uncooked 
round and the second sample lost approximately 15 per cent of the riboflavin when 
compared to the untreated sample. The veal sample that was fried, number k^> } 
could unfortunately not be compared since the untreated sample was not available 
for assay. There was evidently little loss of riboflavin in the fried pork ham 
samples. A sample of smoked ham after frying also showed little or no loss after 
the cooking. Two samples of pork loin showed different results however, for one 
showed practically no loss while the other decreased about 50 per cent. There 
is no apparent explanation for the higher loss in the second sample. It may be 
that different muscle are more greatly affected than others. 

In comparing the values In the table, it should be remembered that a certain 
tissue is listed with its riboflavin content before and after cooking. It will 
be seen that a few samples that were stewed have no parallel control since no 
sample of the untreated meat was available. However, there are a sufficient num- 
ber of samples of a particular meat so that a Justifiable comparison can be made 
between the riboflavin content of a cooked sample and the average riboflavin con- 
tent of the untreated tissue. Several such cases exist in samples that were stew- 
ed. Two samples of stewed heart gave values which are very nearly equal to those 
figures for several of the samples of untreated heart. While this is not entirely 
a fair comparison, the figures obtained for other meats indicate that as a rule 
the average of several untreated tissues can serve as the figure to which the 
cooked samples can be compared. 

A sample of stewed beef kidney showed no decrease from the untreated tissue. 
There was apparently little loss in the beef spleen samples that were stewed, but 
some riboflavin was lost in the samples of beef heart. It appears from the data 
that riboflavin is not easily loat under our conditions of stewing. It is true 
that the period of 3tewing was for a half hour and that the temperature was slight- 
ly below the boiling point of water, and with these conditions it might be expect- 
ed that the loss would be very slight as compared to treatment at higher tempera- 
tures and for a longer time. The size of the meat particles is also a factor in 
retaining much of the riboflavin. 



When samples of pork loin were baked the destruction of riboflavin was as 
great a3 50 per cent. It appears from the table that roasting also causes de- 
struction of riboflavin in excess of 30 per cent. This loss is approximately 
equal to the loss of thiamin. Since the natural pH of meat is approximately h.k 
to k.6 it does not seem very probable that riboflavin would be destroyed, but it 
is quite likely that the effect of leaching is as important here as in the ex- 
planation of the thiamin losses. Since riboflavin occurs in combination with 
compounds of high molecular weight, presumably proteins, it is possible that after 
cooking when the proteins are denatured, the riboflavin is more easily leached 
from the tissue. The increased temperatures and longer cooking time may also aid 
in the loss of riboflavin from the cooked meats. 



106 







TABLE X 




■ 


Riboflavin Content 


of Cooked 


and Processed Meats 




Sample 




Micrograms/ Micrograms/ 


Meat 


number 


Process 


gm. fresh gm. dry 


Beef heart 


87 


Stewed 


12.3 37.0 


Beef heart 


. 5V 


11 


7.0 3^.0 


Beef kidney 


81* 


Dried 


18.7 90.0 


Beef kidney 


85 


Stewed 


90.0 


Beef kidney 


112 


11 


30.2* 85.O 


Beef liver 


.98 


Dried 


100.0 


Beef liver 


99 


Fried 


65.O 


Beef liver 


110 


Dried 


37.0 125.0 


Beef liver 


111 


Fried 


37.8* 86.0 


Beef liver 


120 


Dried 


30.0 100.0 


Beef liver 


121 


Fi'ied 


ho. 6* 95.O 


Beef round 


uo 


Dried 


3.5 11.0 


Beef round 


U2 


Fried 


2.9* 10.0 


Beef round 


105 


Dried 


1.9 7.0 


Beef round 


106 


Fried 


5.0 


Beef round 


107 


Roasted 


1.0* 5.0 


Beef spleen 


88 


Stewed 


l.k 13.0 


Beef spleen 


108 


Dried 


16.0 


Beef spleen 


109 


Stewed 


15.0 


Veal hindquarter 


^ 


Fried 


2.9 11.0 


Pork ham 


li+2 


Roasted 


3.4* 7.2 


Pork ham 


iia 


Fried 


2.69 8.3 


Smoked ham 


^7 


Dried 


2.9 9.0 


Smoked ham 


U3 


Fried 


2.6 3.0 


Pork loin 


36 


Dried 


2.0 . ; 7.0 


Pork loin 


37 


Baked 


.65* 3.0 


Pork loin 


38 


Fried 


l.kk* 6.0 


Pork loin 


92 


Dried 


2.7 9.0 


Pork loin 


93 


Fried 


1.31* fc.'o 


Pork loin' 


9h 


Roasted 


.91* . 3-5 



*Calculated approximation 



. . 107 

Discuss ion of the Results 

These assays verify previous reports that liver, kidney and other animal 
tissues contain high amounts of riboflavin "hut in addition furnish quantitative 
data for a large number of meat products prepared under a variety of conditions. 
Any comparison of the values for riboflavin reported here and those of other 
workers must of necessity be limited only to reports within the most recent years. 
The failure to clearly differentiate between the factors of the "vitamin Bp com- 
plex" has complicated the results obtained "by earlier workers. It is thus desir- 
able to make comparison with those results which have been obtained by the use of 
crystalline riboflavin as the standard. The practice of converting the results 
of experiments of previous years which were based on growth into actual micrograms 
of riboflavin is not completely trustworthy. The growth obtained with crystalline 
riboflavin today using "apparently similar rats and similar diets" is no assur- 
ance that it can justifiably apply to the conditions of previous years and serve 
as a means of placing previous reports on a quantitative basis. The use of fluo- 
rometric methods, rat growth experiments, and microbiological assay procedures 
can be compared with some justification if crystalline riboflavin is used as a 
basis for that comparison. However, it is also necessary to point out that some 
of the rat growth methods employed diets which were not complete in all essential 
nutrients other than riboflavin. This criticism of earlier investigations must 
discredit some reports which have until now been accepted as reliable. 

The greater part of the available figures from the literature on the ribo- 
flavin content of animal tissues is summarized in Table XI. Together with the 
values obtained in this study, it is possible to obtain s.n indication of the vari- 
ation in riboflavin content of many samples and from several species. 

Livers and kidneys of the different species are uniformly higher than the 
other organs in their riboflavin content. The tissues, heart and spleen are ap- 
preciably higher than the muscle. The muscles or beef, pork, veal and lamb con- 
tain riboflavin in approximately the same amount. 

The values in the tables demonstrate the wide variation that must be expected 
when samples from various sources are assayed. The feeding and care of animals 
in different parts of our country or in foreign countries would influence the 
amount of riboflavin in the tissues. There is evidence for this in the reports of 
several workers. The muscle of the chicken is low in riboflavin, but the darker 
muscle contains appreciably more than does the lighter muscle. 

The results reported in thi3 chapter indicate that the riboflavin content of 
meat and meat products is. affected by household as well as by commercial process- 
ing. The losses due to frying and to stewing are not a serious consideration since 
such losses are definitely lower than the losses due to roasting. Although the 
losses are sometimes as high as 50 per cent, the figures obtained clearly show 
that the riboflavin content cf the cooked meats makes this class of foods an im- 
portant source of the vitamin. 

The Ability cf Meat to Supp^ the Riboflavin Requirement 

From the figvre3 available, the average white family of the North Atlantic 
States 3pends $1.38 to $2.'49 per capita weekly for food. The figures for this 
group can be used as the basis for a calculation cf the per capita riboflavin re- 
quirement and for the estimation of the amount of riboflavin furnished by the 
meat in our diet. Assuming that the daily requirement for riboflavin is 1800 
micrograms per day for 3^5 days, the yearly need for this vitamin would be 657 
milligrams. Since U8.5 pounds or 22,000 grams of beef and veal were consumed 



108 

during this time, arid if the beef muscle contained 2.6 micrograms per gram, then' 
beef and veal furnish 57 milligrams. Similar calculations for mutton, lamb and 
pork, indicate that these supply 8 milligrams and 2*+. 2 milligrams respectively. 
The total riboflavin supplied by meat and meat products is at least 90 milligrams 
or one-seventh of the requirement of riboflavin. Making the calculation another 
way, it can be seen that llU pounds of meat is consumed in this food group, and 
using 2.6 micrograms a3 the value for the riboflavin content, this amount of meat 
would supply 135 milligrams of riboflavin which is more than 20 per cent of the 
requirement. This figure includes miscellaneous meat products such as sausages, 
liver, poultry and fish. It is apparent then that meat supplies at least one- 
fourth of the requirements when it is considered that the large amount of ribo- 
flavin furnished by such tissues as liver and kidney is not used in the above cal- 
culations . , 

The ability of meat and meat products to supply the human daily requirement 
can also be ascertained by a more simple calculation. For example, calves liver 
supplies about ?0 micrograms per gram, which means that 60 grams of the uncooked 
tissue would supply the daily requirement. In spite of the slight loss on frying 
this liver, the requirement can be met with less than 100 grams, or one -fourth 
pound of this liver. In the case of muscle meats like beef round or pork loin, 
the cooked meats are able to supply a considerable portion of the daily require- 
ment of this vitamin. Other organs, like heart and spleen, also supply a sizeable 
share of the vitamin. 

The study of Stiebling and Phipard (102) on the diet3 of families of employed 
wage earners and clerical workers in cities, enables one to obtain the proportion 
of riboflavin furnished by the meat in the diet. The study in different sections 
of the country showed regional differences in consumption of various groups of 
foods, the most striking being in respect to separate food, particularly in the 
meat, fish and poultry class. Beef was preferred in most regions. It is inter- 
esting to note from the report of Stiebling and Phipard that about 25 per cent of 
the total amount spent for food was for meat and from the present data this amount 
of meat and meat products furnishes nearly 30 per cent of the riboflavin require- 
ment, assuming the daily need is l800 micrograms p r r day. These workers have con- 
cluded from their wide surveys that as more money is' spent for food the diets are 
correspondingly richer in riboflavin. When the per capita expenditure level was 
$1.88 to $2.^9 per week, an average of nearly 2 milligrams of riboflavin was con- 
'sumed. With the higher expenditure levels the intake was greater and therefore 
in the range where a liberal supply of the vitamin was obtained. Only about 1 pur 
cent of the families studied showed an intake of riboflavin that was inadequate. 
It can be concluded therefore that adequate riboflavin is supplied in the average 
diets of families of white employed workers. 

A more recent publication by Stiebling and her co-workers (ll8) has presented 
data which shows that white farm operators obtain 50 per cent of their riboflavin 
requirement from milk, and l6 to l8 per cent of the riboflavin requirement from 
meat, poultry and fish. The increased consumption of milk by this group of in- 
dividuals is markedly different from that in the group of city workers previously 
studied by Steibling and her co-workers. The high proportion of milk in the diet 
accounts for the greater percentage of the riboflavin requirement which is furnish- 
ed to the farm families. This is in contrast to the lower consumption of milk and 
increased consumption of meat in the diet of white employed wage earners. 



L09 



TABLE XI 
Literature Summary on the Riboflavin Content of Animal Tissues, 



Description 



Micrograms/gm. 
fresh 



Mi cr o gr ams /gm . 

dry 



References 



Muscle Meats 
Lean pork 



Bacon 
Cured ham 
Fresh ham 
Sheep 

Sheep, fat 
Sheep, lean 



2.k 
2.25 
,88-. 91 
.90 

2.0 
3.0 
2.7 

6q 



3.48-3.7U 



3.99 



Sheep, lean 








2.68 


Beef, lean 








1.55-2.23 


tT II 






3.? 




It It 






2.25 




IT II 




1.8 


•1.9 




. 11 " 




X 


1.-.59 




Veal 






3-0 




11 




.69-. 72 




11 








2.81-2.91 


Horse loin 






6.63 




Hcrse loin 








2.21 


Horse leg muscle 






5.U6 


Horse neck muscle 






3.98 


Hcrse neck muscle 




1.14 




Horse, lean- 




1.01 


-1.10 




Horse leg 






1.36 




Chicken 










Composite 


sample 






1.99 


Leg 








3.01 


Breast of 


hen 




.52 


• 9<5 


(red) 






.78 




(white) 






.26 




Breast 








3.0 


Lamb, lean 






.69 


2.68 


Lamb, lean 






2.1 





9k 

75 
82 

9^ 
15 
15 
15 
76 
9^ 
9^- 
9k 
9h 

75 

82 

8 

9k 

75 
9 k 
9k 
9h 
9k 
9k 
9k 
9k 
9k 
9k 

9k 
9k 
9k 
9k 

9k 
k2 
9k 
82 



110 







TABL3 XI (continued) 




Description 


Micrograms/gm. Micrograms/gm. 
fr.esh dry 


References 


Muscle Meats 








Lamb, fat 


1.0 




94 


Lamb chops 


2.8 




.15 


Dog muscle 




1.79 


9k 


Dog muscle 


.59 




Qk 


Rabbi t 


.59 




9>! 


(red) 


.75 




94 


(white) 


M 


> 


94 




10.4-11.9 




46 


Liver 








Pork 


35.1 




75 



Beef 



Calf 



Chicken 
Rabbit 

Sheep 



35.1 






37.0 






35.0 






27.0 






(Fall and 


Winter) 




18.0 






23.0 






19.0 






28.1 






20.0-30.0 






14.6-19.5 




55.0-74.0 


18.0 






10-20.0 






13.0 


(Fall) 




16.0 


(Winter) 




9.0-18.0 






1.0-24.0 






35.6 






20.0 


(Winter) 




16.5 






15.0 


(Fall) 





111 . 



14.5-17.7 
7.17 
6.45 

43.5 (Winter) 
49.5 (Fall) 
16.5 



90 



76 
76 
73 

82 

85 

90 

75 

8 

94 

+ 82 
32 
73 
73 
3^ 
55 

12 

73 
82 

73 
42 

46 
12 
54 

73 
73 
82 



Ill 



TABLE XI (continued) 



Description 



Brain 
Rabbit 

Heart 
Beef 

Babbit 



Lung 
Beef 



Micrograms/gm. 
fresh 



Micrograms/gm. 
dry 



k. 32-5. 18 

3.1 

2.7 



3.5 

9.0 

13.3 
6.15 
5.53 



Chicken 






Kidney 






Beef or 


Veal 


21 . 


Beef 




10.8 

10.0-20.0 
8.0-16.0 


Rabbit 




— -r.> 
13.2 
13.1 


Spleen 






Beef 




.5-1.0 


M 




1.97 


Stomach 






Beef 




.87-1A7 


Rabbit 




27.I 



17.5 



71.2 



10.9 



References 



.5-1.0 
3.3 



22.2 



k€ 
12 
5h 



9h 

9k 
82 

k6 
12 
5k 

k2 



32 + 9k 
9k 

32 

3k 

12 
k6 
55 



32 

9k 



9k 
k6 



32 

9k 



Rabbit 



9.9 
9.89-12.94 

8.9 



12 
k6 
54 



113 



LITERATURE CITED 



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2. Axelrod, A.E., Lipton, M.A. and Elvehjem, C.A. Production of Uncomplicated 
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3. Axelrod, A.E., Sober, H.A. and Elvehjem, C.A. The d -Amino Acid Oxidase 
Content of Rat Tissues in Riboflavin Deficiency. J. Biol. Chem., 13^, 7^9 
(19*0). — 

k. Bender, R.C. and SuiDplee, C-.C. Significance of Quantitative Relationships 
in Vitamin B Complex Studies. J. Am. Chem. Soc, $9, 1178 (.1937). 

3. Bessey, O.A. Vitamin G and Synthetic Riboflavin. J. Nutr., 15, H (1933). 

6. Bethke, R.iM., Record, P.P. and Wilder, O.H.M. Further Studies on Vitamin G 
in Chick Nutrition with Snecial Reference to Flavins. Poul. Sci., l6, 175 

• (1937). , : 

7. Bethke, K.M., Record, P.R. and Wilder, O.H.M. The Effect of the Ration of 
the Sen on the Vitamin G Content of Eggs with Observations on the Distribu- 
tion of Vitamin B and G in Normal Eggs. J. Nutr., 12, 309 (1936). 

3. Baars, J.X. Geneesk. Tijdschr. Nederland. Indie, J_8, 31^5 (1938). (Batavia) . 

9. Booher, L.E., Blodgett, H.M. and Page, J.W. Investigations of the Growth 
Promoting Properties of Vitamin G Concentrates. J. Biol. Chem., 107, 599 
(193*0. 

10. Bourne, M.C. and Pyke, M.A. The Occurrence of Cataract in Rats Fed on Diets 



11. 

12. 

13, 

15. 

16, 
17, 



13, 



Deficient in Vitamin 3 2 . Biochem. J. 23, 1865 (1935) 

Bourquin, A. and Sherman, H.C. Quantitative Determination of Vitamin G. 
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Charite, A.J. and Khaustov, N.W. Flavin Content of Animal Tissues under 
Different Conditions. Biochem. J., 29, J>h (1935). 

Cohen, F.H. Eine Objektive Methode Zur. Bestimmung der Fluorescenz mit 
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Cohen, F.H. Vitamin B 2 Bestimmung Mittels Me s sung der Fluorescenz. Arch. 
Neerland de Physiol., 20, 167 (1935). 



Darby, W.J. and Day, P.L. Riboflavin Content of Meats 
(1938). 



J. Nutr., 16, 209 



Day, P.L. Vitamin G in Beef and Veal. J. Home Econ., _23, 657 (1931). 

Day, P.L. Vitamin G Deficiency. Am. J. Public Health, 2k, 603 (193 J 0. 

Day, P.L., Darby, W.J. and Langs ton, W.C. The Identity of Flavin with the 
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11)+ 

19. Day, P.L. and Langston, W.C. Further Experiments with Cataract in Albino 
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20. Day, P.L., Langs ton, W.C. and Cosgrove, K.W. The Appearance of Cataract and 
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21. Day, P.L., Langston, W.C. and O'Brien, C.S. Cataract and Other Ocular 
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22. Donelson, E.G. and Macy, Icie, G. Human Milk Studies: The Vitamin B and 
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23. Ellinger, P. Lyochromes in' the Kidney with a Note on the Quantitative Estima- 
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26. Elvehjem, C.A. and Koehn, Jr., C.J. Studies on Vitamin B 2 (G) - Non Identity 
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115 



35- Fraser, H.F., Topping, II. E. and Isbell, E. The Bacterial Assay of Riboflavin 
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116 

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ol. Kuhn, R., Kaltschmitt, H. and Wagner-Jauregg, T. Ueber den Flavingehalt 

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71. Kuhn, R. and Weygand, E. Synthetisches Vitamin B 2 . Ber. d. Deutsch. Chem. 
Gesell., 67, 208J+ (193*+). 



117 

72. Laazt, L. and Verzar, F. Hemmung dea Wachs turns Durch Jodeasigaaure und 
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in the Rumen of the Cow. J. Biol. Chem., 133, lxv (1940). 

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»V 



118 

90. Pulver, R. Analytische Bestimmung dea "Freien Flavins" Durch Dialyse und 
Ultrafiltration. Zeit. Vitaminforsch., 10, 88 (19*K>). 

91. Richardson, L.R. and Hogan, A.G. Skin Leaions of the Rat Associated with 
the Vitamin B Complex. Res. Bull. 2*H, Missouri Agr. Exper. Sta. (1936). 

92. Rose, M.S. Laboratory Handbook for Dietetics. Uth Ed., (1937) MacMillan 
Co . , Hew York . 

93- Saffry, O.G., Cox, H.S., Kunerth, B.L. and Kramer, M.M. A Biological Asaay 
of Riboflavin in Liver of Cow, Calf, Sheen, Lamb and Hog. J. Nutr., _20, 169 
(191+0). 

9k. Schormuller, J. Uber daa Vorkommen von Vitamin Bg ( Lac to flavin ) . Zeit. f. 
Untera. des. Lebenam., 77, 1, ?6k (1939). 

95- Sebrell, W.H; and Butler, R.E. Riboflavin Deficiency in Man. A Preliminary 
Note. Public Health Reports, r 53, 2282 (1938). 

96. Sebrell, W.H. and Onatott, R.H. Riboflavin Deficiency in Doga. Public 
Health Reports, 53, 83 (1938). . 

97- Sherman, H.C. and Lanford, C.S. Riboflavin, Dietary Sources and Requirements 
The Vitamins, A Symposium, 1939- Am. Med. Assoc. 

98. Snell, E.E. and Strong, F.M. A Microbiological Assay for Riboflavin. Ind. 
and Eng. Chem.,Anal. Ed. 11, 3^6 (,1939). 

99. Spies, T.D., Vilter, R.W. and Ashe, W.F. Pellagra, Beriberi and Riboflavin 
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100. Stem, K.G. Potentiometric Study of Photof lavins . Biochem. J., 28, 9U9 
(193*0. 

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(1939). 



119 



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h 

112. Warburg, 0. and Christian, W. Ueber ein Neues Oxydat ions ferment und sein 
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U.S. Dept. Agr., 1941. 



CHAPTER XI 
NICOTINIC ACID 

Page 
History of Nicotinic Acid 121 

Chemistry of Nicotinic Acid 122 

Physiology of Nicotinic Acid 12U 

Pharmacological Properties of Nicotinic Acid 125 

Nicotinic Acid Therapeutics 125 

Biological Assay Methods for Nicotinic Acid 126 

Chemical Methods for Determining Nicotinic Acid 127 

Microbiological Methods for the Determination of 

Nicotinic Acid 128 

Biological Assays of Meats for Nicotinic Acid 129 

Results of Biological Assays for Nicotinic Acid 130 

Chemical Analysis of Meats 132 

Results of Chemical Analysis for Nicotinic Acid 132 

Microbiological Assays of Meat for Nicotinic Acid 1J>6 

Discussion of Nicotinic Acid Results 136 

Literature Cited __ 1^3 



CHAPTER XT 
NICOTINIC ACID 

History of Nicotinic Acid 

The discovery of a new organic compound was made by Huber (Ul,U2) in 1867 
through the chance oxidation of nicotine with potassium dichromate and sulfuric 
acid. He regarded it as pyridinecarboxylic acid, but it remained for Wei del 
(110) to name it nicotinic acid. Later, Veidel (ill) showed that the oxidation 
of ,3picoline also gave this same compound, and it was thus shown that nicotinic 
acid is £ pyridine carboxylic acid. The first Isolation of nicotinic acid from 
a natural material was accomplished by Suzuki et al (98) from a concentrate of 
rice bran. Funk (28) and Drummond and Funk (19) corroborated this finding, and 
Funk later found nicotinic acid in yeast and in his "crystalline vitamin frac- 
tion". Since the vitamin fraction had beriberi preventative ability, it was 
natural for Funk to say that "it is not unlikely that nicotinic acid is a decom- 
position product of the vitamin". It is understandable then, that nicotinic acid 
was first proposed as the anti beriberi factor since the acid was isolated from 
rice bran. When the compound was fed to pigeons, however, there was no cure of 
the typical head retraction in the polyneuritic birds, although some workers at- 
tributed a "sustaining action" to nicotinic acid since it seemed to delay death 
of the pigeons maintained on s polished rice diet. It soon became clear that 
nicotinic acid was net the anti beriberi vitamin. Only after many years was nico- 
tinic acid again in the picture when Vickery (105) found nicotinic acid in surface 
yeast, and Warburg and Christian (107) and von Euler et al (21) demonstrated that 
the amide of nicotinic acid was present in the coferments, coenzymes I and II. 
At this time Kuhn and Vetter (58) isolated nicotinamide from a vitamin B concen- 
trate of ox heart, von Euler and Malmberg (22) attributed significance to their 
findings that rats required nicotinic acid for their growth. At present there 
seems to be 3ome disagreement with this conclusion on the basis of studies of 
other workers. Enight (U6) found the compound to be necessary for the growth 
of staphylococci on a synthetic medium, and Mueller (69,70) isolated the substance 
from liver. At this time Funk and Funk (29) claimed that nicotinic acid had a 
stimulating effect on the appetite and prevented loss of weight in pigeons and 
rats. 

For many years, Goldberger and hi3 co-workers at the United States Public 
Health Service had attempted to find the cure for the disease pellagra. After 
several theories were proposed for the endemic condition, Goldberger and his co- 
workers claimed that the diet played a great part in the onset of the disease. 
The recognition that the condition in dogs known variously as black tongue, dog 
typhus, or Southern canine plague, was similar to human pellagra stimulated in- 
tensive laboratory experimentation on the disease. Cary (12) also pointed out 
the similarities of the Chittenden -Underhi 11 syndrome (lU) and the condition, of 
black tongue. Although diet wa3 the causative factor in pellagra as suggested 
by Goldberger, Waring, and Willets (31) it remained for Spencer (91) to point 
out that there were great similarities between spontaneous black tongue in dogs 
and pellagra in human3. It remained for Wheeler, Goldberger and a southern 
veterinarian, Blackstock (112) to recognize that the cases of black tongue in 
the household dogs in certain regions in the South always occurred simultaneously 
with a complaint of pellagra in some member of that household. These observations 
were the basis for the experiments which extended the evidence that the disease 
in dogs and in man were similar and were in fact due to a deficiency in the type 
of food consumed. 



•121- 



122 

Several laboratories became interested in the isolation of the factor that 
would prevent the condition of black tongue in dogs. The fractionation work on 
liver which had been carried out at the University of Wisconsin for a number of 
years had given definite indications that some material in liver could cure or 
prevent the condition in dogs (1+9,50). Further work (20) finally culminated in 
the demonstration that nicotinic acid would cure and prevent the typical symptoms 
of black tongue. It appears advisable to try the chemical compound in the treat- 
ment of cases of human pellagra, and Fouts et al (25), Spies et al (9*0, Harris 
and Hassan (38), and Smith and co-workers proclaimed that the compound was indeed 
the antipellagra factor (88). Thus nicotinic acid again assumed an important 
role in the nutrition of higher animals and the work accomplished at the beginning 
of the twentieth century served as the link between the naturally occurring com- 
pounds and their physiological activity. 

Chemistry of Nicotinic Acid - 

Nicotinic Acid is a fairly simple chemical compound having a slightly acid 
taste. The melting point is between 235° and 237° C The compound is not hygro- 
scopic, and it is stable in air. Nicotinic acid will sublime, and it occurs as 
a white powder or in the form of needles when crystallized from either alcohol 
or water. 

Nicotinic acid is soluble in water and ethyl alcohol to a slight extent but 
much more soluble in hot solvents. As part of the original work to develop a 
cyanogen bromide method for determining nicotinic acid by the authors, it was 
necessary to ascertain the solubility of the compound in a number of laboratory 
solvents. This was done since it seemed advisable to extract the nicotinic acid 
away from the interfering materials with which it occurs in natural products. 
The solubility trials in this laboratory using various solvents resulted in the 
data given in table XII . 

The table on solubility shows that water is a good solvent for nicotinic 
acid. Ethyl alcohol is nearly as good as water, but the higher alcohols, amyl, 
butyl, and isobutyl show less ability to dissolve the substance. Acetone shows 
only moderate ability to dissolve the acid. In general it appears that most 
organic solvents are not good for dissolving nicotinic acid, and that mixtures of 
solvents are not better than the individual ones. Sandier (6) has given, some 
figures on the solubility of nicotinic acid, and the Merck brochure (7*0 on the 
physiological activity and clinical use of nicotinic acid has listed some of the 
solubility figures. There appears to be some discrepancies in the literature 
on the solubility of nicotinic acid in organic solvents. Eandier (6) lists nico- 
tinic acid as being soluble in 85O parts of ether, while the Merck publication 
asserts that "it is insoluble in ether". 

The oustanding chemical property of nicotinic acid is its stability to most 
chemical reagents. It is made by the nitric and chromic acid oxidation of nico- 
tine, and few organic chemical compounds will withstand this reaction. It is also 
very stable to alkali, but in the evaporation of an aqueous solution or an acetone 
solution there is considerable loss (6). Nicotinic acid amide is much more sol- 
uble in water, alcohol, and acetone than is the free acid. Its physiological 
activity is similar to that of nicotinic acid, and more of these properties will 
be described in a following section. The chemical formulae of nicotinic acid 
and nicotinamide are given below. 



123 



H 



HC 



^ 



\ 



T3-C-0-0-H 



HC CH 



rs 



HC 1 - C - C - - N H, 



HC CH 

y ■ 






CgH^OgN 
Nicotinic Acid 



C 6 H 6 0N 2 



Nicotinic Acid Amide 



TABLE XII 

Solubility of Nicotini c Acid in Various Solvents 
at 22° to 25°C* (67) 



Solvent 



Grams Nicotinic Acid 
Soluble in 100 Grams 
of the Saturated Solution 



Water 
Acetone 
75$ Acetone 
Ether (Ethyl) 
Ethyl Alcohol 
Ether -Alcohol (50$) 
Isobutanol 
Amyl Alcohol 
Isoamyl Alcohol 
Benzene 
Toluene 
Xylene 

-Petroleum Ether A 
Petroleum Ether 3 . 
Chloroform 
Carbon Tetrachloride 
Dioxane 
Pyridine 
Ethyl Acetate 



1.639 
0.281 
1 . 119 
' 0.175 
1.558 
1.362 
0.508 
.O.678 

. H93 

0.232 
0.000 
0.008 
0.000 
. c 00 
0.0k6Q 

0.0153 
0.351 
10.03 
0.117 



*The majority of this data, was gathered by Dr. Olaf MickeJsen 



124 

Physiolog3 r of Nicotinic Acid 

The identification of nicotinic acid as the pellagra preventative vitamin 
was further basis for the speculation regarding the physiological function of 
nicotinic acid. The importance of nicotinic acid in the oxidation-reduction re- 
actions involving carbohydrate metabolism had received attention by the work of 
several European investigators. The suggestion by von Euler and co-workers (21, 
22) that nicotinic acid might be part of the respiratory ferment, cozymase, re- 
sulted in the identification and isolation of nicotinamide by Warburg and 
Christian (109) as already cited. Nicotinic acid has since been shown to exist 
in practically all animal tissues. Both coenzymes I and II, take part in the 
transfer of hydrogen which is essential to the chain of reactions by which intra- 
cellular oxidation is accomplished. As soon as the significance of nicotinic 
acid was recognized, attempts were made to relate the physiological function of 
this compound to coenzymes I and II which were already established as important 
links in the chain of carbohydrate metabolism. A logical study, therefore, was 
the determination of the coenzymes in normal body tissues and in nicotinic acid 
deficient animals. This has been done by von Euler and co-workers (2k) with rats 
maintained on a totally vitamin B complex deficient diet. It was found that the 
analyses of the avitaminotic rat tissues contained less cozymase than the tissues 
of normal rats. The administration of nicotinic acid to the deficient rats 
brought the cozymase content up to the normal level. 

The work of von Euler and his co-workers has been criticized because the de- 
termination of cozymase was done on the rat. Since it has not been conclusively 
shown that nicotinic acid is required by the rat, it would appear more advisable 
to use the tissues of the nicotinic acid deficient dog or pig for the determina- 
tion of coenzyme I. Such a study was made by Axelrod, Madden, and Elvehjem (3). 
It would found that the cozymase content of the same tissues taken from both the 
pig and the dog fell within a rather limited range. In animals suffering from a 
nicotinic acid deficiency, however, there were marked changes in some of the tis- 
sues. No decrease in the blood cozymase of severely nicotinic acid deficient dogs 
or pigs was demonstrated. The liver and muscle tissue from deficient dogs con- 
tained considerably less cozymase and, therefore, probably less nicotinic acid 
than the same tissues from normal dogs. No such differences, however, could be 
demonstrated in the brain or in the kidney. 

A further approach to this problem ha3 been the study made by Kohn, Klein 
and Dann (53). These workers used the growth of the organism, Hemophilus para - 
influenzae , as the criterion of the V factor content of the black tongue tissues. 
Since the V factor is considered to be a combination of coenzymes I and II (di- 
phosphopyridine nucleotide and triphosphopyridine nucleotide) the method depends 
upon the quantity of extract necessary to produce that amount of bacterial growth 
equal to that produced by a standard. Although the method does not distinguish 
between diphosphopyridine nucleotide and triphosphopyridine nucleotide the re- 
sults of this assay procedure are expressed as "coenzyme-like substances". Kohn, 
Klein, and Dann found that the V factor content of liver and of striated muscle 
from black tongue dogs showed significant variation from that found in normal 
dogs. No changes were noted in the heart, pancreas, kidney, or brain. These 
workers conclude that no evidence was found which would indicate that black tongue 
results from a generalized decrease in the coenzyme level of the tissues. The 
determination of the V factor in the urine and tissues of black tongue dogs has 
been made by Pittman and Fraser (j6) using a similar organism similar. to that of 
Kohn, Klein, and Dann. It was found that the V factor content of certain tissues 
of the dogs could be correlated to the intake of nicotinic acid. The liver, muscle 
and heart showed the greatest changes of any of the tissues studied. Another 
study by Dann and Kohn (l6) using their same technic indicated that the levels 
of the V factor in the tissues of rats getting nicotinic acid did not differ from 



the tissues of rats which were not getting nicotinic acid, 
that the coenzymes were being synthesized "by the rats. 



125 

These workers showed 



Some insight into the probable metabolism of nicotinic acid was obtained 
by Axelrod, Spies, and Elvehjem (k) in their studies on the coenzyme I content 
of blood and muscle of pellagrins. These workers found that the coenzyme I con- 
tent of erythrocytes did not decrease significantly in pellagra, but the coenzyme 
content of the muscle did show a definite lowering. They also found that pyra- 
zine monocarboxylic acid, coramine, and quinolinic acid did not effect the in 
vitro synthesis of coenzyme I. 

The blood V factor content of normal and pathological subjects was studied 
by Kohn, Bernheim, and Felsovanyi (51) who concluded that the coenzyme content 
of the blood of pellagrins was in the lower portion of the normal range of values, 
Other pathological states showed a certain amount of variation. The lowest 
values occurred in diabetics, while the highest values were found in patients 
with pulmonary disease. Axelrod, Gordon, and Elvehjem (2) have shown that large 
amounts of nicotinic acid, given to normal people will increase the coenzyme I 
level of the blood. On the basis of their studies these workers concluded that 
no diagnostic value can be attributed to determinations of the coenzyme I level 
of the blood of suspected borderline nutritional cases. 

An interesting observation on the in vitro synthesis of factor V in human 
red blood cells has been made by Kohn and Klein (52). They found a 65 per cent 
increase in the cells after incubation with nicotinic acid. Confirmatory evi- 
dence was also obtained by Axelrod, Gordon, and ElA^ehjem (2) in their in vivo 
work . 



Pharmacological Properties of Nicotinic Acid 

The importance of nicotinic acid as a vitamin led to some toxicological and 
pharmacological observations by a few workers. Chen, Rose, and Robbins (13) 
found the minimal lethal dose for mice to be U5OO milligrams per kilo body weight 
and 3500 milligrams for guinea pigs and rats. The administration of 2 grams of 
the acid daily to monkeys resulted in diarrhea, loss of weight, convulsions and 
death within three weeks. Tomita and collaborators (101) produced paralysis in 
the extremities of pigeons and fowls by injecting 0. 5 to 1.0 grams of the sodium 
salt into the birds. Unna (102) found that the toxic effects are due in part to 
the acidity of the substance. Kon and Funk (5*0 claimed that nicotinic acid 
caused a rise in the blood sugar of rabbits. Spies et al (9*0 noted the effect 
of administering nicotinic acid to normal men. They found dilatation of the 
3mall blood vessels of the skin, especially in the extremities. These workers 
found that dosages. from 300-1500 milligrams orally, or about 20-100 times the 
daily requirement, caused some slight discomfort in their patients. 

Nicotinic Acid Therapeutics 

Nicotinic acid has received considerable attention within recent years be- 
cause of its dramatic role in the cure of many of the classical symptoms that 
have been associated with the endemic disease pellagra. It has now been accepted 
by the clinician as a definite aid in the treatment of endemic pellagra. Spies, 
Bean, and Ashe (93) have presented a review of nicotinic acid therapy and discuss 
its successful use in the treatment of acute and chronic cases. Many of the 
fulminating cases show a dermatitis, diarrhea and deme.itia, with concurring 
degrees of diseases of the 3kin, gastrointestinal tract, and nervous system. 
Characteristic glossitis and stomati'tr's often occur in pellagrins. Achylia • 
gastrica, involvement of many mucous membranes, and impaired nervous function 



126 

have "been noted in this disease. Although the role of nicotinic acid in the 
treatment of pellagra is a most important one, the inadequate diet usually con- 
sumed by these patients complicates the picture so that it i3 necessary to admin- 
ister other vitamins. The typical symmetrical skin lesions and the glossitis 
and stomatitis have "been cured by giving nicotinic acid. Other symptoms some- 
times develop together with a complete loss of many sensory perceptions and de- 
creased nervous coordination (86). In severely pellagric patients the adminis- 
tration of nicotinic acid seemed to bring about a typical riboflavin deficiency. 
This was obviously due to the fact that the patients were nutritionally deficient 
not only in nicotinic acid, but in the other members of the vitamin B complex as 
well. Similar indications of multiple deficiencies were obtained when both nico- 
tinic acid and riboflavin were administered to patients of poor nutritional his- 
tory. The presence of a thiamin deficiency became apparent after the symptoms 
of riboflavin and nicotinic acid avitaminoses were cleared up. The use of nico- 
tinic acid in pellagra ably demonstrated that some of the psychotic symptoms 
could be cured at the same time as the cutaneous and oral lesions. This led to 
the experiments by a number of workers who used the compound in attempts to clear 
up several related symptoms. Cleckley, Sydenstricker, and Geeslin (15) found 
that stupor of atypical psychotic states could be prevented by the administration 
of nicotinic acid. The auditory acuity was sharpened by such treatment. Vin- 
cent's Angina has been said to have been successfully treated with nicotinic acid 
(J+5) • A large number of cases having the encephalopathy syndrome with the cloud- 
ing of consciousness, uncontrollable gasping and sucking reflexes have been 
treated with good results with nicotinic acid (k-j) . Improvement has been noted 
when nicotinic acid therapy has been employed in cases of Meniere's syndrome (39), 
lead poisoning symptoms, high tone deafness, lupus erythematosus, the edema of 
nephrosis, chronic ulcerative colitis, and multiple sclerosis (68). The use of 
nicotinic acid in counteracting the untoward symptoms of sulfanilamide adminis- 
tration has resulted in relief of nausea, diarrhea, confusion, dizziness, head- 
ache, and general malaise (l8). It has also received some attention in prevent- 
ing roentgen ray sickness and porphyrinuria (92). 

Biological Assay Methods for Nicotinic Acid 

In the period 1925 to 1930 several papers appeared in the Public Health 
Reports by Goldberger and his co-workers in which the anti-blacktongue potency 
of various foods was given by feeding a great many foodstuffs to dogs which were 
fed the experimental diet consisting largely of corn but which contained some 
cowpeas, salts and fat. This work was primarily that of Goldberger, Wheeler, 
Lillie, Rogers, Hunt, Tanner and Sebrell. A typical report on this method and 
the results was published in 1928 (33). A summary of much of the assays done by 
this method has been written by Sebrell (85). 

This dog assay method was then the best approach for the appraisal of the 
pellagra preventative factor in biological materials until 1939. Although the 
knowledge of the vitamins has progressed since the work of Goldberger and his co- 
workers, the value of this assay method was markedly increased when it was found 
that nicotinic acid was the pellagra preventative factor. The use of crystalline 
nicotinic acid furnished a standard with which a fairly quantitative estimation 
of nicotinic acid in foodstuffs could be obtained. 

The use of dogs for' bioassay purposes has the advantage of furnishing infor- 
mation as to the actual nicotinic acid potency of a particular foodstuff, regard- 
less of the form in which nicotinic acid occurs. In this way, the actual utiliza- 
tion of the nicotinic acid present will be reflected in the cure of symptoms and 
the response in growth. The method which we used is essentially that of Goldberg- 
er and his co-workers with the added precautions of furnishing many of the crystal- 
line vitamins that may have been absent or present in inadequate amounts In the 
Goldberger ration. 



127 

The experiments of McKibbin and associates (60,61,62) on the use of purely- 
synthetic diets for dogs may furnish another means of producing experimental 
"black tongue. The diet consists of sucrose, cottonseed oil, purified casein, 
salts, and the available synthetic vitamins, other than nicotinic acid. Typical 
symptoms of black tongue can be obtained on such a diet with the advantage that 
the length of time necessary to deplete the dog of its nicotinic acid reserves 
is appreciably reduced. 

Chemical Methods for Determining Nicotinic Acid 

The identification of nicotinic acid as the anti -pellagra factor stimulated 
work to perfect chemical methods that would allow accurate estimations of this 
important compound. The chemical methods of analysis for nicotinic acid have 
been patterned primarily after those which have been used for the determination 
of pyridine. The greater chemical reactivity of the pyridine ring as compared 
to the benzene ring has enabled certain reactions to occur. The nitrogen atom 
in the ring has been' singled out as the position at which the ring can be broken 
down, and the proposed methods for the determination of nicotinic acid depend on 
the breakdown of the pyridine nucleus, the opening of the ring, and the suitable 
conjugation of the free ends with an aromatic amine. .... 

A number of compounds have been used to react with the tertiary nitrogen 
for the purpose of splitting the pyridine ring, vbngerichten, (105) and Reitzen- 
stein (79) first proposed 2,H, dinitrochlorobenzene as the compound which by 
adding to the nitrogen would weaken the linkage so that the ring would open. 
Konig (55) used cyanogen bromide, phosphorus trichloride, and other compounds in 
his attempts to open the ring. Once the nitrogen has its greatest, valence satis- 
fied l>y the addition of any one of these compounds, the ring is more susceptible 
to decomposition. The ring can be easily broken with the liberation of the nitro- 
gen in the form of a tertiary amine or the ring can be opened without the loss 
of any atom. The carbon chain thus remaining combines with the aromatic amine 
to give a colored compound. The carbon chain is a derivative of glutaconic 
aldehyde, which probably enolizes to give a reactive hydroxyl which can combine 
with the organic base. The function of the cyanogen bromide is to open the ring 
and the aromatic amine to combine with the free ends. A complete discussion on 
the chemistry of the reaction has been published by Waisman and Elvehjem (106). 

Swaminathan (99) first recommended the use of cyanogen bromide in the reac- 
tion and used aniline as the organic base. Shaw and McDonald (87) applied the 
cyanogen bromide method to liver extracts and obtained fair results. They pointed 
out the danger of absorbing out interfering colors with charcoal since nicotinic 
acid will also be absorbed. A note on the reliability of the cyanogen bromide 
method has been given by Kringstad and Naess (56) in which they point out the 
effect of pH on the reaction. In a subsequent publication Kringstad and Naess 
(57) presented a complete study of the effect of pH, time, light, and other con- 
ditions that would affect the specificity of the cyanogen bromide -aniline reac- 
tion. 



Melnick and Field (63) have offered the important procedure of completely 
clarifying the unknown solution so that a satisfactory blank can be obtained. 
By the use of a special charcoal in an acid alcohol solution the nicotinic acid 
is allowed to pass through unabsorbed thus yielding a water white solution in 
which the color can be. developed and easily read. Melnick, Robinson, and Field 
(6U) have contributed their findings on the study of pyridine compounds in the 
urine which react with the reagents to give a color similar to that obtained 
with nicotinic acid. These workers have given further evidence (65) for the 
urinary excretion of nicotinic acid and its derivatives by normal individuals. 
Still another paner by these workers (66) gives some evidence. for the factors ' 



128 

affecting the concentration and distribution of nicotinic acid in the blood. 
Swaminathan has also applied his cyanogen bromide method to a study of the urin- 
ary excretion of nicotinic acid under a variety of dietary conditions (100). 

Yilter, Spies, and Mathews (10U) suggested a method based on 2,U dinitro- 
chlorobenzene addition followed by reaction with sodium hydroxide. The purple- 
red color formed could be read and the quantity of nicotinic acid measured within 
the limits of 0.1 and 1.0 milligram. The method has the disadvantages that it 
is not readily applicable to biological materials, is not specific and will give 
only semi -quantitative results. Karrer and Keller (kh) have also used the 2,k 
dinitrochlorobenzene reagent for their reaction. These workers have given quan- 
titative values for some animal tissues using their method but it is obvious that 
these values are much too low since actual amounts of nicotinic acid isolated 
from equivalent liver or liver extract are much higher than that reported by these 
workers . 

Numerous attempts have been made to apply the nicotinic acid methods to the 
determination in urine and blood. Invariably, the authors cited above have indi- 
cated that their method can be applied to both blood and urine. There are a num- 
ber of reports which question these statements, one of which is by Schindel (8*0 
who claims that creatinine yields a color with dinitrochlorobenzene. Perlzweig, 
Levy, and Sarett (72) have determined nicotinic acid derivatives in human urine. 
They have given figures for trigonelline excretion under different conditions and 
have discussed the rise in trigonelline after the ingestion of nicotinic acid. 
The investigations on the quantity of nicotinic acid in urine have furnished 
further modifications of the method by Bandier (7) and Harris and Raymond (^0). 
Roos (8l) has used a Stufenphotometer in his measurements of the color. Kodicek 
(k"J f k8) has increased the sensitivity of the method so that it would be more 
widely applied. This worker points out the necessity of differentiating between 
"true r ' and "apparent" nicotinic acid, von Euler, Schlenk, Heiwinkel, and Hogberg 
(23) describe their modification of the methods. Bandier and Hald (9) have given 
an exact colorimetric method of analysis using p-methyl amjnophenol sulfate as 
their reagent. Porje (77) used the color reaction for urine studies. Del Regno 
(78) has described a further modification of the nicotinic acid method. Bandier 
has presented nicotinic acid values of biological materials as obtained with his 
modification of the method (6). Ritsert (80) has given explicit directions in 
the use of organic reagents in the extraction of the nicotinic acid from bio- 
logical materials, but the disadvantage of having incomplete extraction of the 
nicotinic acid from aqueous solutions has limited its use. The method has been 
followed in this laboratory with unsatisfactory results. 

Pearson (71) has presented a simple method for the determination of nicotin- 
ic acid in blood. Briggs (ll) and Friedman and Barborka (27) have also applied 
the cyanogen bromide reaction to the determination of nicotinic acid in blood. 
Rosenblum and Jolliffe (82) have reported a modification of Bandier and Hald's 
method for the determination in urine. 

Microbiological Methods for the Determination of Nicotinic Acid 

There are a number of microbiological methods that have been proposed for 
the determination of nicotinic acid. One of these has been used by Fraser, 
Topping, and Sebrell (26) with some success in urine samples. The organism which 
they have used is Shigella paradysenteria (sonne). Dorfman, Horwitt, Koser, and 
Saunders (17) have used the dysentery bacillus which is grown in a synthetic 
medium. Rubinstein and Shekun (83) have intimated that the wax moth Galleria 
melonella can be cultivated so that a definite response can be obtained with as 
little as 1 microgram of nicotinic acid. Pittman (75) has used the parainfluenza 



129 

organism to determine the so-called V factor, and Lvoff and Querido (59) have 
indicated that Proteus required nicotinic acid so that this organism can also be 
used for bioassay purposes. A recent method proposed "by Snell and Wright (90) 
for the microbiological determination of nicotinic acid is patterned after the 
riboflavin assay of Snell and Strong (89). The organism that is used is the 
Lactobacillus arabinosus and it is grown on a suitably free nicotinic acid medium 
for three days. The lactic acid produced by the organism is titrated with stan- 
dard alkali. A series of nicotinic acid levels furnish the calibration curve by 
which the results can be calculated. Application of this method to meat and 
meat products will be mentioned later. 

Biological Assays of Meats for Nicotinic Acid 

The diet used throughout the experiments with dogs was a modification of the 
Goldberger diet (32). The corn, cowpeas, and cottonseed oil diet is satisfactory 
for the production of experimental black tongue, but because of the difficulty 
in getting a uniform supply of California cowpeas, together with the fact that 
several of the crystalline vitamins were available to make the diet more complete 
in the known factors, it was felt that a simpler ration would be more satisfac- 
tory for a large series of assays. The ration which was used in all the assays 
had the following composition: 



Ground yellow corn 


71 


Purified casein 


18 


Salts 


k ! " 


Cottonseed oil 


5 


Cod liver oil 


2 


Thiamin 


50 micro grams /kilo body weight/ day 


Riboflavin 


cr\ M II II II II 


Pyridoxine 


t-(-\ II M II II II 


Halibut liver oil 


2 drops twice weekly 



The purified casein was prepared by suspending crude casein in fifty times 
its weight of water. The mixture was stirred, allowed to settle, and the super- 
natant water drained off. This washing and decantation was repeated until the 
casein was washed six times. As a final step in the purification, the washed 
casein was dissolved in alkali and reprecipitated by the careful addition of 
hydrochloric acid. The supernatant was again drained and the casein washed once 
more. It was dried in a current of warm air and ground in a burr mill. The salt 
mixture was that of Phillips and Hart (73) with the MnS0^-7H20 increased from 
0.7 to 10.0 grams per kilogram of salt mixture. The diet was prepared in 10 kilo- 
gram lots and kept in the cold room when not in use. The vitamin solutions were 
fed by rubber -tipped pipette to the dogs daily. The thiamin, riboflavin and py- 
ridoxine were given at levels that were shown to be sufficient for the dog as 
judged by other experiments in this laboratory. They were all fed at 50 micro- 
grams per kilogram body weight of the dog per day after the daily weighing. It 
was believed that sufficient pantothenic acid was supplied by the corn. An 
averaged sized dog of 7.5 kilograms would consume about 35 grams of ration per 
kilogram body weight per day, and since a pantothenic acid assay gave 9 micrograms 
per gram of yellow corn, it is apparent that a dog consuming about 300 grams of 
ration would be getting 1890 micrograms of pantothenic acid per day. This figure 
calculated in terms of body weight would mean that approximately 250 micrograms 
of pantothenic acid per kilogram of body weight was supplied. Although no figures 
are available on the pantothenic acid requirements of dogs, there was indirect 
evidence that the modified Goldberger diet contained sufficient of this factor 
for the dog so that it was not a complicating factor in the accuracy of the 
assays . 



130 

Mongrel dogs were used in all the bioassays. The animals were obtained 
shortly after weaning and were placed on a diet of dog food and milk for a week 
or 10 days. Near the close of this preliminary period the dog food was gradually 
decreased in the diet and replaced by the modified Goldberger diet. When the 
animal was accustomed to the corn diet, the milk was also gradually removed so . 
that no difficulty was encountered in making the animal consume the dry ration. 
The dogs were weighed daily and carefully examined for any symptoms of black 
tongue . 

The dogs began to show the, typical black tongue symptoms within one to three 
months. There occurred a gradual plateau in the weight curve during this period, 
followed by beginning signs of anorexia and lassitude. At times the symptoms 
became aggravated until the full blown condition of black tongue was apparent. 
The glossitis and stomatitis were accompanied by the foul odor of the breath. 
The increased whiteness of the gingiva was due in all probability to the Vincents 
organism. When the first distinct drop in weight was observed, the dogs were 
standardized by giving a definite amount of crystalline nicotinic acid in a cap- 
sule. The quantity of nicotinic acid usually used was 20 milligrams. The dogs 
responded to the vitamin within 12 to 2k hours by beginning to eat the ration. 
There was a progressive gain in weight until in 5 to 1^ days the restoration of 
weight had reached a peak, and within lk to 21 days the dogs had again lost weight 
and reached the level at which they wer j standardized. The response in weight 
to the standard dosage of nicotinic acid was noted and the gain in weight per 
milligram of nicotinic acid was calculated. A foodstuff to be assayed was then 
fed to the individual dog at a level which would contain very nearly the same 
amount of nicotinic acid as the standardizing dosage of the vitamin. 

It was frequently observed that a dog on the deficient diet would have re- 
peated attacks of black tongue with a decreasing amount of time elapsing between 
the attacks. This apparent decrease in the animals' efficiency to respond to the 
crystalline vitamin was believed due to gastro -intestinal difficulties. Some 
indication of the fact that certain animals had a decreasing ability to respond 
to a supplement was obtained in a few animals and the assays in these cases were 
discarded. In order to minimize any error due to variation in the animal, each 
dog was restandardized after two successive assays by again feeding 20 milligrams 
of nicotinic acid in a capsule. To increase the accuracy of the assays a food- 
stuff was usually fed to more than one dog and was reassayed on the same dog at 
a later period. The sample of meat that was to be fed to the dog was fed in 
three equal portions suspended in a small amount of water within 2k hours. These 
portions were avidly consumed by the dog. 

Results of Biological Assays for Nicotinic Acid 

The nicotinic acid values of the 35 meat samples available for biological 
■assay are given in Table XIII. The figures are given for both the dried tissue 
and the fresh meat. Liver is an outstanding source of the vitamin as shown 
earlier by Goldberger and co-workers (30). Our results verify the earlier quali- 
tative observations and in addition give more complete and accurate values on a 
■large number of meat products. Although several samples of particular tissues 
vary in their nicotinic acid content, many of the samples gave uniform results. 
Pork, beef, -veal, and lamb liver are uniformly higher than any other tissues 
assayed. The samples of beef kidney and pork kidney were next in potency. The 
samples cf veal hindquarter showed a surprisingly high content of nicotinic acid 
with the range of values eqi^al to that found in the kidney samples. The samples 
of pork heart and beef heart were nert in order of potency and were closely 
followed by samples of spleen and the muscular tissues of pork. In general the 
muscular tissues varied in their nicotinic acid content. The tissues such as 
lung, pancreas, brain and tongue showed wide variation in nicotinic acid content. 



:able xi i: 



131 



Nicotinic Acid Content of Meat and Meat Products 
Determined "by Biological Assay with Dogs 

Milligrams of Nicotinic Acid 
Per 100 grams of Tissue 



Tissue 



Sample 






Number 


Dry Weight 


Fresh Weight 


33 


90.0 


26.1+ 


122 


110.0 


27.5 


110 


110.0 


27.5 


120 


85.O 


25.0 


121 


87.O 


29.1+ 


70 


72.0 


22.5 


61 


131.0 


1+6.0 


96 


135.0 


39.2 


62 


72.0 


15.5 


8U 


81.0 


16.9 


126 


89.0 


17.8 


71 


32.0 


8.0 


73 


23.0 


!+.9 


76 


28.0 


7.0 


108 


33.0 


8.3 


123 


52.0 


12.3 


95 


1+0.0 


9.7 


32 


38.0 


10 . k- 


114 


37.0 


10.0 : 


113 


32.0 


8.8 


115 


15. ° 


5.2 


116 


28.0 


8.2 


119 


•' 25.0 


8.3 


lh 


^6.0 


13.0 


92 . 


25.0 


7.5 


125 


19.0 


5.3 


82 


1+6.0 


12.8 


75 


70.0 


16.1 


kh 


72.0 


18.0 


103 


2I+.0 


6.5 


77 


30.0 


7.5 


Uo 


15.0 


3.8 


105 


3^.0 


-. ' 10.2 


107 


37.0 


10.2 


79 


11.0 


2.7 


6^ 


16.0 


3.5 


78 


33.0 


8.3 



Pork liver 
Pork liver 
Beef liver 
Beef liver 
Beef liver, fried 
Veal liver 
Lamb liver 
Lamb liver 
Pork kidney 
Beef kidney . 
Beef kidney 
Pork heart 
Beef heart 
Beef spleen 
Beef spleen 
Beef spleen 
Pork ham 
Pork ham 
Pork ham 
Pork ham 
Boiled ham 
Smoked ham 
Tenderized ham 
Pork loin 
Pork loin 
Pork loin 
Eeef tongue 
Veal hindquarter 
Veal hindquarter 
Veal hindquarter 
Beef "brain 
Beef muscle 
Beef muscle 
Roast beef 
Beef pancreas 
Beef pancreas 
Beef lung 



132 

The effect of processing, "both household and commercial, on the nicotinic 
acid content of meats was negligible. The sample of beef liver which was fried 
contained the same amount of nicotinic acid as the untreated sample of liver. 
The sample of beef round that was roasted showed no loss in the potency of nico- 
tinic acid. A single sample of boiled ham showed approximately half the nicotinic 
acid in untreated samples of pork ham. This I03S is undoubtedly due to the ex- 
traction of the nicotinic acid from the water in which the meat was cooked. 'The 
smoking and tenderizing processes had little effect since the values obtained 
were very nearly the same as those values for the untreated tissues. It was to 
be expected that the losses of nicotinic acid due to cooking would be at a mini- 
mum since the compound is very stable; any losses that were obtained are explained 
by the partial washing out of the nicotinic acid present in the tissues as free 
acid or as one of the nicotinic acid containing coenzymes. 

Chemical Analysis of Meats 



The chemical method that was employed in the analysis of meats and meat 
products was a modification of a method proposed by Melnick and Field (63). The 
use of the preferential charcoal adsorption step aided in the clarification of 
the material to be analyzed so that the color reaction could be carried out with 
the greatest convenience. The exact procedure which was followed in the deter- 
mination of animal products has been described in a previous publication by 
Waisman and Elvehjem (106). The procedure outlined is readily adapted to animal 
tissues, but the analyses of plant products is beset by several difficulties. 
The main difficultjr encountered in the analysis of plant materials has been at- 
tributed to interfering alkaloids having the pyridine nucleus. 

In order to obtain the most reproducible results, 0.1 gram samples of dried 
liver and kidney and 0.25 gram samples of the various dried muscles were used for 
analysis. The sample wa3 transferred to a 15 ml. graduated centrifuge tube, 5 
ml. of water added, followed by 5 ml. of concentrated HCT. The tube was placed 
in a hot water bath for ^5 minutes, cooled, made up to 15 ml. before transferring 
to a flask containing 200 milligrams of a special charcoal (Darco). Ten ml. of 
absolute alcohol were added, the flask shaken and filtered through quantitative 
filter paper, into a dry flask. Eight and thirty-three one -hundredths ml. ali- 
quots were placed in clean centrifuge tubes, a drop of phenolphthalien added, 
and sufficient saturated WaOH added dropwise, until a faint color of pink appear- 
ed. The solution was brought back to neutral with split drops of HC1 using an 
outside indicator brom thymolblue or nitrazine paper. The volume at this stage 
must be below 10 ml. Three ml. aliquots of the neutralized filtrate were taken 
for the development of color with the cyanogen bromide and aniline. The manipu- 
lative details of this procedure are found in a previous paper (106) or in the 
article by Melnick and Field (63). The decolorization procedure with the special 
charcoal yielded water white solutions as a rule. Very slight color was obtained 
with the liver samples, due in part to the great concentration of bile and liver 
pigments, but this did not give any significant interference with the blank read- 
ing. In some cases it was advisable to use 50 additional milligrams of the char- 
coal for complete decolorization. 

Results of Chemical Analysis for Nicotinic Acid 

It was the purpose of the chemical analyses to check the values obtained by 
the dog bioassay on the same samples. Unfortunately some of the samples were not 
available at the time that the chemical determinations were made, but a suffici- 
ently large number of samples were determined chemically to furnish some basis 
for comparison between the two methods. A total of about 200 analyses were made 
on over fifty samples of the dried tissues and the values are given in Table XIV. 



I 



133 



TABLE XIV 
Nicotinic Acid Values Obtained "by Chemical 







Analyses o 


f Animal Tissues 

Milligrams of N 
per 100 grams 


Icotinic Acid 
of tissue 






Sample 








Tissue 


Number 


Dry Weight 


Fresh Weight 


Beef 


liver 


98 


69.I 


21.0* 


Fried beef liver 


121 


55.5 


15.8 


Beef 


liver 


131 


77.8 


22.7 


Beef 


liver 


150 


58.6 


16.1 


Beef 


liver 


152 


51.2 


15.1 


Veal 


liver 


70 


50.3 


13.2 


Veal 


liver 


'97 


69.5 


20.2 


Veal 


liver 


158 


72.1 


19.5 


Pork 


liver 


122 


9^. 5 


29.8 


Pork 


liver 


153 


Go.h 


18.7 


Pork 


liver 


1-59 


7lU 


20.0 


Lamb 


liver 


96 


59.1 


17.2 


Beef 


kidney 


81 


56.2 


8.26 


Beef 


kidney 


126 


32.1 


6.42 


Stewed beef kidney 


112 


1U.7 


5.23 


Pork 


kidney 


62 


47.9 


10.5 


Pork 


kidney 


83 


45.4 


9.86 


Pork 


kidney 


137 


45.0 


9.05 


Stewed beef heart 


51+ 


15.9 


3.3 


Stewed beef heart 


87 


24.4 


7.32 


Beef 


heart 


155 


42.4 


8.15 


Beef 


heart 


ikj 


37.0 


7.03 


Veal 


heart 


155 


50.7 


10.6 


Beef 


spleen 


76 


25.3 


6.2* 


Stewed beef spleen 


109 


21.1 


6.3* 


Beef 


spleen 


130 


38.8 


8.15 



13* 



TABLE XIV (Continued) 



Milligrams of Nicotinic Acid 
per 100 grama of tissue 





Sample 






Tissue 


Number 


Dry Weight 


Fresh Weight 


Fried "beef round 


1+2 


3^.1 


9.05 


Beef muscle 


105 


21.5 


6.39 


Roast round of beef 


107 


20.0 


4.28 


Beef Round 


128 


37.0 


8.7 


Beef round 


l*+5 


30.2 


7.7 


Broiled beef round 


lVj 7 


22.5 


— 


Fried beef round 


11+8 


19.2 


— 


Beef round 


151. 


33.0 


8.2 


Beef round 


151+ 


27.8 


6.87 


Beef round 


157 


27.9 


7.8 


Fried veal steak 


^5 


28.9 


8.38 


Veal hindquarter 


103 


33.0 


8.9 


Veal hindquarter 


129 


36. h 


9.05 


Veal hindquarter 


13^ 


31. k 


6.8 


Lamb leg 


80 


32.7 


8.1+8 


Pork ham 


32 - 


20.3 


5.56 


Fried pork ham 


52 


ik.k 


3.7 


Pork ham 


95 


27.0 


6.6 


Pork ham 


12U 


35.0 


8.26 


Pork ham 


139 


33.0 


8.35 


Smoked ham 


^7 


25.2 


8.2 


Fried amoked ham 


1+8 


30.2 


11+.3 


Smoked ham 


102 


19.5 


5.55 


Smoked ham 


116 


13.1 


3.86 


Tender ham 


72 


l*k0 


1+.1+ 


Tender ham 


117 


15.3 


5.27 


Tender ham 


119 


23.0 


7.66 


Boiled ham 


115 


19.3 


6.7 


Boiled ham 


101 


18.3 


5.95 



135 



TABLE XIV (Continued) 



Milligrams of Nicotinic Acid 
per 100 grams of tissue' 



Tissue 


Sample 
Number 


Dry Weight 


Fresh Weight 


Pork loin 


36 


21.2 


6.62 


Broiled pork loin 


37 


20.8 


U.l+5 


Fried pork loin 


38 


. 23.2 


5.57 


Fork loin 


lh 


21.U 


6.05. 


Pork loin 


125 


22.2 


6.1 


Pork loin 


156 


22.2 


.6.66 . 


Beef pancreas 


6k 


18.0 


5.84 


Beef Tongue 


32 


22.0 


6.12 


Beef "brain 


132 




5.05 


Beef "brain 


i^k 


22.2 


' M 


Beef lung 


138 


30,h 


\ 6.18 


Light Chicken 


68 


29.7 


7.3 


Dark chicken 


66 


23.8 


6.1k 


Cod muscle 


135 


13.5 


2.3 


Salmon muscle 


136 


26.4 


7.35 



■Calculated Approximation 



A 



136 

It appeared from the chemical analysis that the livers from the various 
species were indeed the richest sources of nicotinic acid of all the tissues 
studied. The kidney, spleen and heart were also in the same order of potency 
as found "by the "biological assay. The chemical analyses of the muscular samples 
showed the same variation in nicotinic acid content aa was observed in the 
values obtained by the biological assay. The tissues, lung, pancreas, brain, 
and tongue were all about in the same range. 

Several processed samples which wore analyzed by the chemical procedure 
showed only slight changes in nicotinic acid content. The samples of pork loin 
which were fried and broiled compared favorably with the untreated sample of 
loin. .A number of commercially processed hams showed slightly lower values than 
comparable tissues which were untreated. Two out of the three smoked ham samples 
showed lower values than samples of pork ham or pork loin, but the third smoked 
ham sample contained approximately the same quantity of nicotinic acid as the 
untreated samples. Several of the stewed tissues were analyzed but could not be 
compared to the untreated tissues. 

Microbiological Assays of Meats for Nicotinic Acid 

Some, preliminary assays for nicotinic acid by the microbiological technic 
of Snell and Wright (90) were performed on several samples of meat. Some of 
these samples were analyzed by the biological dog method and by the chemical 
test. Although Snell and Wright's method involves the use of autolysis for the 
liberation. of nicotinic acid from the animal tissues studied, the ready applica- 
tion of their method to dried samples of meats has not been fully investigated. 
The extraction of the nicotinic acid from the dried meat samples was done by two 
procedures. The meat sample was suspended in water and autoclaved with both acid 
and alkali. No significant differences in nicotinic acid content was observed 
in the two types of extraction. It still remains to be determined whether an 
enzymatic digest would give more complete liberation of the nicotinic acid than 
the alkaline. extractions, and for this reason the microbiological values given 
below must be considered as preliminary. 

The assay is performed by suspending a sample of the dried meat in water 
and adding sufficient alkali to make it 8 per cent NaOH. The suspension is 
neutralized and a suitable aliquot is taken for analysis. The organism, 
L actobacillus arabin osus is grown on a nicotinic acid free basal medium which 
consists of acid hydrolyzed casein, tryptophane, cystine, glucose, sodium acetate 
and a variety of growth factors which have been shown to be essential for this 
particular group of organisms. The preliminary values obtained by this new 
microbiological procedure are given in Table XV. 

The order of nicotinic acid potency in the animal tisaues found by the two 
previous methods has been substantiated by the results of the microbiological 
method. Liver is uniformly higher than the other tissues followed closely by 
kidney. The spleen and muscular tissues follow liver and kidney in nicotinic 
acid content. 

Discussion of Nicotinic Acid Results 

In general the values obtained by the methods described in previous sections 
are in the same range. An examination of Table XVI shows that the biological 
assays with dogs gave the highest values, while lower values were obtained by 
the chemical analyses and microbiological assay. It will be observed that the 
greatest differences are found in those tissues which contain the largest amount 
of nicotinic acid. A possible explanation for these differences lies in the 



137 



TABLE XV 
Nicotinic Acid Content of Meats 



Determined fry the Mj cr ofriological Method* 



Sample 



Sample 
Number 



Milligrams Nicotinic Acid 
per 100 grams dry sample 



3eef liver 
Fried "beef liver 
Pork liver 
Beef liver 
Veal liver 
Lamb liver 
Beef kidney 
Pork kidney 
Beef spleen 
Steved "beef spleen 
Beef snleen 
Fried pork ham 
Tender ham 
Boiled ham 
Boiled ham 
Smoked ham 
Pork loin 
Light chicken 
Beef pancreas 



131 

121 

122 

98 

70 

96 

126 

62 

I30 

109 

76 

lUl 

119 

101 

115' 

102 
lh 
68 
6k 



1*7.6 

38.0 

•5H.0 

hk.Q 

1*2.0 

32.0 

lk.0 
I5..8 

20.0 
1^.0 : 
16.0 
12.7 
1U.2 

li*.5 

: 23.5 
29.0 

15.7 



^Assays made fry L. J. Teply 






138 

fact that liver contains several substances which stimulate metabolism in the 
dog and will result in growth not due to nicotinic acid alone. Conversely the 
large amount of muscular tissue which must "be fed in the biological assay to the 
deficient dog may present difficulties of absorption thus decreasing the accuracy 
of the assay. 

The best agreement in the various samples was observed in the results of 
the chemical determination and microbiological assay. Samples of kidney, ham, 
and veal muscle showed nearly identical results by the two methods. Further 
work now in progress on the microbiological assay of meats may narrow the differ- 
ences observed in some tissues. 

The high nicotinic acid potency of liver has been verified by both chemical 
analysis and bioassay. Kidney must be considered as good as most samples of 
liver. The difference in species does not seem to affect the nicotinic acid 
content of these tissues, for the same order of potency holds for the organs of 
the hog and ox. Beef spleen must be placed high in the list of nicotinic acid 
rich tissues with muscle meats next in order and followed closely by heart and 
tongue. These last two tissues are muscular and have values ranging in the 
neighborhood of skeletal muscle. Other organs, such as lung, brain, etc., are at 
the end of the list but should not be considered low sources. They are definitely 
higher than most cereals, milk, and grains on the dry basis. The high content 
of nicotinic acid in liver, kidney, and spleen can be accounted for by their 
high metabolic activity. The occurrence of nicotinic acid in the red blood cells 
perhaps accounts for the high value in the spleen. The liver and kidney help to 
elaborate the enzymes and coenzymes of which nicotinic acid is an important part, 
thus furnishing the rationale for the higher values obtained in these tissues. 
The liver undoubtedly stores nicotinic acid and this in part accounts for the 
values found. 

Nicotinic acid is known to be one of the most stable of the vitamins and the 
losses due to cooking should be significantly less than the other vitamins of the 
water soluble group. It is, therefore, interesting to note that some losses were 
observed in both commercial and household cooked meats. 

The values that were obtained by the biological and chemical methods should 
be compared to those obtained by other investigators using other chemical pro- 
cedures. The available literature on the nicotinic acid content of animal tissues 
has been summarized in Table XVII. It should be kept in mind that the values are 
on different animals in various parts of the world where the state of nutrition 
evidently played a part in the vitamin content of the tissues. In general how- 
ever, the range of values obtained by us and by other investigators is approxi- 
mately the same. Bacharach (5) has recently summarized the available figures on 
the distribution of nicotinic acid in human and in animal foods. 

It is worth while to mention in a quantitative manner the ability of meat 
to furnish a large part, if not all, of the daily human requirement of this 
vitamin. The Food and Nutrition Committee of the National Research Council has 
tentatively set the daily human need for nicotinic acid at 10 to 20 milligrams. 
Approximately 15 milligrams of nicotinic acid per day would be required by the 
normal 70 kilogram adult. The amount of meat necessary to supply this require- 
ment would be 100 grams of fried liver or 200 grams of cooked veal chops or beef 
round steak. 









139 



TABLE XVI 
Comparison of Nicotinic Acid Values 



Obtained "by Three Methods 



Milligrams of Nicotinic Acid per 
100 grams of dried tissue 



Tissue 


Sample 


Biological 


Chemical 


Microbiological 




Number 


Assay 


Analysis 


Assay 


Pork liver 


122 


110.0 


9>.5 


54. 


Fried "beef li Tr er 


121 


87.0 


53-3 


38.0 


Beef liver 


131 


-- 


77.3 


1+7.0 


Veal liver 


70 


72.0 


50.8 


1+1.0 


Beef liver 


98 


-- 


69.1 


1+1+.0 


Perk kidney 


62 


72.0 


1+7.9 


45.5 


Beef kidney 


126 


-- 


32.1 


32.0 


Beef spleen 


76 


28.0 


23.3 


20.0 


Pork ham 


95 


1+0.0 


27.0 


28.0 


Boiled ham 


115 


15.0 


19.3 


11+.2 


Tenderized ham 


119 


25.0 


23.0 


16.2 


Pork loin 


74 


1+6.0 


21.1+ 


23.5 


Veal hindquarter 


103 


2lK 


33.0 


33.2 


Veal hindquarter 


13* 


-- 


31.4 


29.0 


Beef pancreas 


6k 


15.0 


18.0 


15.7 


Light chicken 


68 


-- 


29.7 


29.0 



140 

There is another way of calculating the proportion of our nicotinic acid 
requirement which is furnished by the meat we eat. If we take the figures of 
Stiebling and Phipard (96) it is seen that 139 pounds of meat, poultry, and fish 
were consumed per capita by the inhabitants of the North atlantic states in 1935- 
Subtracting the 20.9 pounds of fish and other sea food and the 16.2 pounds of 
poultry there remains 102 pounds of meat and meat products. This calculates to 
0.28 pounds or 127 grams of meat per capita per day. Assuming that this is all 
muscle in the form of beef, veal, lamb, or pork, and that 100 grams of the cooked 
meat contained about 8 milligrams, this would mean that over two-thirds of the 
nicotinic acid requirement would be furnished by the meat. Some of the meat is 
undoubtedly consumed in the form of liver, heart, and spleen which furnish more 
nicotinic acid than the muscular tissues of veal, beef, or pork. The same calcu- 
lation can be made for other cross sectional areas described by StieblJng and 
Phipard, and the same large percentage of the daily requirement for nicotinic 
acid is supplied by the meat of these consumers. Still another means of calcu- 
lating the amount of nicotinic acid furnished by the meat consumed can be arrived 
at from the proportion of the various food constituents which furnish the caloric 
intake necessary. Calculations such as these also point out the fact that animal 
tissues are able to furnish the larger share of our daily need for nicotinic 
acid. 



TABLE JCVII 

Literature Summary of Nicotinic A c Id 

Content of Animal Tissues 



Tissue 



Milligrams per 
100 grams fresh 
Weisfht 



References 






Beef liver 
Ox liver 
Beef liver 
Ox liver 
Beef liver 
Ox liver I 
Ox liver II 
Ox liver III 
Beef liver 
Sheep liver 
Sheep liver 
Pork liver 
Pork liver 
Rabbit liver 
Rabbit liver 
Horse liver 



18.6 

17.0 

9.2 

12.2 

12.9 

18.-19.5 

18.7-20.0 

15.5 

9.3 

12.5 

20. C 

11.8 

9.6 

Ik. 3 

7.85 
16.0 



hi 

^7 

113 

7 

1 

57 
57 
57 
kk 

99 

^7 

7 

1 

6 

80 



in 



Tissue 



TABLE XVII (Continued) 

Milligrams per 
100 grams fresh 
Weight 



References 



Ox muscle 
Ox muscle 
Beef muscle 
Ox muscle 
Beef muscle 
Beef muscle 
Beef muscle 



k.O 

k.3 

2.U 

k.9 

5.7 

k.9 

3.83 



^7 

^7 

113 

7 

1 

,57 
kk 



Pork muscle 
Pig muscle 
Rabbit muscle 
Rabbit muscle 
Horse muscle 
Cod muscle 



3.3 

' fc.73 

1.1.0 

6.5 

k.66 

2.0 



57 

7 

6 

80 

kk 

6 



Ox heart 
Sheep heart 
Pork heart 
Rabbit heart 



5.93 
6.0 

5.3^ 
3.k 



7 

*7 

7 
8o 



Ox kidney-cortex 
Ox kidney -medulla 
Beef kidney 
Sheep kidney 
Pig kidney 



6.56 
5.2 
19.i+ 

7.5 
6.8 



7 

7 

kk 

kl 

7 



Ox adrenal cortex 
Ox adrenal medulla 
Sheep adrenals 



6.5^ 
k.9 
-3.5 



7 

7 

^7 



Sheep pancreas 
Pig pancreas 



k.O 

5.0 



^7 
7 



1U2 



TABLE XVII (Continued) 



Tissue 



Milligrams per 


References 


100 


grams fresh 
Weight 






5-3 


V7 




^.U2 


7 




U.OU 


7 




M 


^7 




0.9 


8o 




3.0 


^7 




1.2 


80 




l*.U2 


7 




3.8^ 


7 




3.0 


7 




1.59 


7 




3.25 


7 



Ox spleen 
Ox spleen 
Pig spleen 

Ox lung 
Rabbit lung 
Ox train 
Rabbit brain 
Fig testes 
Pig ovary 
Ox thyroid 
Pig thyroid 
Pig thymus 






110 



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5- 
6. 

7- 

8. 

9. 

10. 



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80 (1933) 






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li*9 

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CHAPTER XII 

PYRIDOXINE (VITAMIN Bg) 

Page 
Recognition and Establishment of Vitamin B5 151 

Identification, Chemistry and Synthesis of 

Pyridoxine : : 151 

Role of Pyridoxine in Nutrition 152 

Toxicology and Pathology of Pyridoxine Deficiency 15^ 

Methods of Assay 155 

Biological Rat Assay Method for Estimating 

Pyridoxine 155 

Influence of Processing on the Pyridoxine Content 

of Meat 157 

Discussion of Pyridoxine Assays 157 

Literature Cited l6l 



CHAPTER XII 
PYRIDQXINE (VITAMIN Bg) 

Recognition and Establishment of Vitamin B g 

The symptoms associated -with the condition in rats known as "rat pellagra" 
were described "by Gyorgy (27, 30). The deficiency manifested itself as a 
dermatitis with accompanying erythema of the paws, nose, and ears. Gyorgy called 
the factor which prevented this condition "vitamin Bg" and differentiated it from 
the dermatitis caused "by a lack of riboflavin (30). Fractionation studies had 
shown that the factor was absorbed on fullers earth similar to riboflavin, but 
the distinction was made between the florid type of dermatitis due to the defi- 
ciency of vitamin Bg and the denudation and bloody areas of the skin which 
occurred in a riboflavin deficiency. Still further investigation by Birch, 
Gyorgy, and Harris (k) and Lepkovsky, Jukes, and Krause (62) gave evidence that 
the anti -black tongue factor in dogs or its counterpart in human pellagra was 
different from lactoflavin and the "rat pellagra" factor called vitamin Bg. It 
is apparent that the vitamin H of Booher (5) ana the vitamin H of Hogan (kj, hk) 
are identical with vitamin Bg. The factor Y of Chick and Copping (10) is also 
considered to be identical with this vitamin. 



Identification, Chemistry and Synthesis of Pyridoxine 

The final isolation of the vitamin in the crystalline state was accomplished 
in 1938 by several groups of workers, namely, Lepkovsky (6l) Gyorgy (31 )/ Kuhn 
and Wendt (57), Ichiba and Michi (V7), and Keresztesy and Stevens (52). The 
molecular formula of the compound was soon forthcoming and Stiller, Keresztesy 
and Stevens (87) showed that the compound was 2 -methyl, 3 -hydroxy-^, 5 dihydroxy- 
methyl pyridine. Further proof cf this structure was obtained by Harris and 
Folkers (36). The independent work by Kuhn and his coworkers (58, 59, 60) 
corroborated these findings. 

Several papers have appeared on the properties and reaction of vitamin Bg. 
One of these is by Harris (35) in which the acetyl derivatives, dibromides, and 
catalytic reduction products are described. The property of tautomerism is 
described by Harris, Webb, and Folkers (38). The substance is very soluble in 
water, but less soluble in 95 per cent alcohol, in ether, in chloroform, and 
in fats. The hydrochloride of the vitamin is more stable than the free base. 
In cold alcohol 1.1 grams of the hydrochloride is soluble in 100 cc, while 1.5 
grams dissolve in 100 cc. of 95 per cent ethyl alcohol at 70° C. The aqueous 
solution of the vitamin is neutral, but the pH of an aqueous solution of the 
vitamin Bg hydrochloride is nearly 3. A solution of the substance will decompose 
on exposure to light (29, 70). The formulae of the free vitamin and its hydro- 
chloride are given here: 

CH 2 0H 



H0-C 



H 5 G 



CE^OE 
1 2 




j. 


- CH 2 0H 
-H 



x> 



X> 



HO — C 



E^C-C^ 



'N- 



Vitamin Bg 



/ 1J \ 



C-CHgOH 

,, C— H 



H CI 
Vitamin Bg Hydrochloride 



■151- 



152 

Role of Pyr J doxine In Nutr ition 

With the availability of crystalline vitamin B5 it was possible to devise 
experiments which would further the understanding of the function and action of 
pyridoxine in the animal organism. Salmon (7^, 75) found that certain oils 
alleviated the erythema and acrodynia of a typical vitamin Bg deficiency. Quack- 
entmsh and Steenhock (71) found that the unsaponifiahle fraction of wheat germ 
oil had no antiacrodynia potency, and that the esters prepared from the saponi- 
fiable fraction contained the entire activity. Birch (3) presented evidence 
that there exists a relation between vitamin B5 and the unsaturated fatty acids. 
Tange (91) submitted data which suggested that an intimate relationship existed 
between vitamin Bg and the fatty acids as indicated by the production and cure 
of the acrodynia-like dermatitis. This work has received confirmation in the 
results of Schneider, Steenbock, and Platz (76) who claimed that the acrodynia 
can be cured by two different means, the essential fatty acids and the vitamin 
as obtained from a purified rice bran concentrate. Burr, Brown, Kass, and 
Lundberg (7) emphasized the fact that the unsaturated fatty acids should now be 
considered as individual substances in experiments designed to test the effect 
of the unsaturated fatty acids upon vitamin B5 deficiency. Salmon (75) has 
indicated the role of these unsaturated fatty acids in vitamin B^ deficiency. 

The varied symptomatology associated with pyridoxine deficiency is further 
shown by the work of Chick, El Sadr, and Warden (ll) in which they describe a 
fit-like convulsion of the animals maintained on a vitamin Bg deficient diet for 
a prolonged period. Another instance of the action of the vitamin is described 
by Emerson and Evans (19) in which they found that reproductive physiology of 
rats in vitamin B5 deficiency is considerably decreased. No matings were ob- 
served in the deficient rats and the accessory organs of reproduction were found 
to be atrophied. 

Richter, Holt, Barelare, and Hawks (72) have shown that there is a definite 
change in the appetite for fat when animals are placed on a diet low in the 
vitamin B complex. The craving for fat was increased at the height of the 
deficiency, but the intake of carbohydrate and protein was decreased. This evi- 
dently emphasizes the need for extra fat and the decreased demand for protein 
during the complex deficiency and bears out the work of Conger and Elvehjem (13), 
who found that increased protein aggravated the vitamin B5 deficiency. This 
substantiated the work of Richter, Holt, Barelare and Hawks in which it was 
shown that rats on the self selection diet had marked aversion to protein when 
certain factors of the B complex were omitted. 

The outstanding symptom associated with a vitamin B5 deficiency is the lack 
of growth. Yon Euler and Malmberg (20) identified the growth promoting factor 
and the dermatitis healing factor in their rat experiments as vitamin B5. Kuhn 
and Wendt (55, 56) found that increasing amounts of vitamin B5 caused an increase 
in weight of rats kept on a vitamin Bg-free diet. Dimick and Schreffler (15) 
also found that 10 micrograms of pyridoxine satisfied the rats' requirements for 
this vitamin, as indicated by the optimum growth response. 

The role of vitamin B5 in the nutrition of the chick is not as clearly 
defined as in the rat. Hegsted, Oleson, Elvehjem, ; and Hart (39, ^0) were able 
to show that 30 micrograms of crystalline pyridoxine per chick per day was able 
to produce a marked improvement in growth on a vitamin 35 "low diet. Carter and 
O'Brien (8, 9) also found that this vitamin is required by chicks. Jukes (^9) 
has claimed that one of the symptoms of vitamin B5 deficiency in chicks is "ner- 
vous convulsive movements" of the birds and the inefficient utilization of food. 
Fruton, Irving, and Bergmann (25) have clearly differentiated between the factor U 



153 

and pyridoxine requirements of chicks. Since "both factors have similar properties 
in that they are absorbed on fullers earth and are water soluble, the possibility 
did exist that the two factors were identical. However, these workers have sug- 
gested that on the "basis of differences in distribution and on the basis that 
vitamin Bg alone will not give an optimum response the two factors are different. 
During work on the Goldberger diet, it was shown in this laboratory by Waisman 
and Elvehjem (98) that vitamin Bg alone did not increase the growth of chicks, 
but when added to a liver fraction which contained additional factors, a response 
in growth was observed. The available information at present indicates that 
yellow corn contains sufficient vitamin Bg for the chick when fed at 70 per cent 
of the diet. 

In nutritional experiments with dogs, Fouts, Helmer, Lepkovsky and Jukes 
(22) first showed that the microcytic hypochromic anemia that developed in pup- 
pies on a synthetic diet could be prevented or cured by the. administration of 
crystalline vitamin Bg or concentrates of the vitamin (23, 2U). Borson and 
Mettier (6) also obtained relief of hypochromic anemia in dogs with synthetic 
vitamin Bg. These workers attributed some significance to the filtrate factors 
which remained after exhaustive absorption with fullers earth since the hemo- 
globin did not rise to the optimum level with pyridoxine alone. The work of 
McKibbin, Madden, Black and Elvehjem (67) demonstrated that more vitamin Bg is 
necessary to protect against the microcytic hypochromic anemia than is required 
to give optimum growth. 

The need for vitamin Bg in the pig was implied by the work of Chick, MaCrae, 
Martin, and Martin (12), but ruminants evidently do not need this factor as shown 
by the work of McElroy and Goss (61+) and Wegnery 'Booth, Elyehjem, and. Hart (99). 
The need for vitamin Bg is not limited to animals, since it has been definitely 
proven that excised tomato roots (73) require this factor. Vitamin Bg functions 
as an activator for root formation in cuttings and not as a root growth factor 
(h2). 



Investigations on the growth requirements of bacteria and yeasts were given 
great impetus by the animal experiments which demonstrated the indispensability of 
vitamin Bg. A variety of organisms were shown to require the factor for their 
normal growth. Moller (68) first demonstrated that vitamin Bg was necessary for 
the growth of lactic acid bacteria. ' Hutchings and Woolley (^6, 102) used vitamin 
Bg as a growth factor for hemolytic streptococci in a synthetic medium. Mcllwain 
(66) showed a need for vitamin Bg in the medium of the same organism. Koser, 
Dorfman, and Saunders (53) however, found that the dysentery bacillus did not 
require pyridoxin for its growth on a synthetic medium. Vilter and Spies (96) 
found vitamin Bg to be an accessory growth factor for S taphylococcus albus . A 
number of workers have shown that various strains of yeast require this vitamin 
(16, 17, 78). 

The first report that man required this vitamin was given by Spies, Bean 
and Ashe (8U). A study of four patients who had been treated with nicotinic acid 
and thiamin for the pellagra and beriberi symptoms were completely relieved of the 
further symptoms of nervousness, insomnia, irritability and weakness when 50 
milligrams of pyridoxine were given. This work was substantiated by the later 
work of Spies and Associates (85). Urinary excretion studies indicated that 
patients with a history of malnutrition excreted low amounts of vitamin B6 after 
an intravenous injection (86). The condition known as cheilosis has been success- 
fully treated with vitamin Bg according to the work of Smith and Martin (83) but 
this vas disputed by the work of Sebrell and associates (69, 82), who found cures 
of cheilosis with riboflavin in vitamin deficient patients. It may be that both 
riboflavin and pyridoxine are concerned in cheilosis. Kark and associates (50) 
studied the blood response of patients having a microcytic hypochromic anemia and 



154 

found no improvement with large dosages': of. vitamin Bg. Vilter and coworkers 
(95) found that the vitamin gave subjective relief to patients with macrocytic 
anemia and pernicious anemia. A slight reticulocytosis also was observed in 
these patients. Vitamin Bg therapy has "been suggested in the treatment of 
.pseudohypertrophic muscular dystrophy (l), and several publications have 
asserted that patients with Parkinsonism can be successfully treated with vitamin 

b 6 m } 85). 

A recent report on the U3e of pyridoxine in the treatment of Parkinsonism 
has been presented by Zeligs (103). Thi3 investigator found that repeated intra- 
venous dosages of 50 to 100 milligrams of pyridoxine hydrochloride to patients 
with Parkinsonism had no beneficial effect whatever on the course of the disease. 
Another recent report (21) also reported that pyridoxine was of no value in 
treating muscular dystrophy or amyotrophic lateral sclerosis. 

Toxicology and Pathology of Pyrid oxine Deficiency . . 

The toxicity and pharmocological properties of vitamin B5 have been studied 
by Unna and Antopol (9*0 and Unna (92, 93). It was found that the vitamin is a 
substance of very low toxicity, and only excessively large doses, about 3 grams 
per kilogram body weight, produced convulsions and death. The toxicity studies 
were carried out on rat3, monkeys, dogs, cats and rabbits. 

The pathology of vitamin Bg deficiency exemplifies itself in a number of 
ways, as evidenced by the work of Antopol and Unna (2). These workers found upon 
histological examination of the skin, that there were changes in the various cell 
layers, resulting usually in a hyperkeratosis in the stratum granulosa and stratum 
lucidum. There is evidence (33) that a condition of "fatty livers" occurs in 
rats having a vitamin Bg deficiency, and this possibility is further supported by 
the work of Gyorgy and Goldblatt (32) in which they showed perenchymatous fatty 
degeneration, focal and massive necrosis, hyperemia and hemorrhage, together with 
some perilobular fibrosis. McElroy and Goss (65) found a condition of "ringed 
tails" in rats maintained on a vitamin B5 depletion diet. Gross and microscopic 
studies of the ears, paws, nose and other areas of the head by Sullivan and 
Nicholls (88) indicated that abscesses occurred in those areas adjacent to the 
areas of specific dermatitis. There was partial atrophy of the sebaceous glands. 

A number of chemical methods for the determination of pyridoxine have been 
proposed. Kuhn and Low (5*+) claimed it was possible to determine quantities of 
0.02 to 0.08 milligrams of the vitamin through reaction with the Folin-Denis 
reagent. It was found that a one per cent solution of lithium hydroxide gave 
better results than the usual sodium carbonate solution. A preliminary note by 
Scudi, Koones and Keresztesy (8l) intimated that a colorimetric procedure could 
be used for the determination of vitamin B5 in the urine, which involved the use: 
of the phenol indophenol reagents of Gibbs (26). Scudi and coworkers (80) gave 
a short presentation of their method. Scudi, Bastedo and Webb (79) described the 
formation of a vitamin B^-borate complex. .■;. ■ ; , . , , 



o 



The chemical .estimation of vitamin B5 in foods by means of the diazo reaction 
and the phenol reagent was described l>y Swamlnathan (89, 90). The diazo reaction 
involves the simple condensation of the diazotized sulfanilic acid with the vita- 
min. The phenol reaction was similar to that referred to by Kuhn and Low. 



155 



Methods of Assay 



A number of "biological assay methods which. U3e rats ad the experimental 
animal have "been suggested by Lunde and Kringstad (63 ), Schneider, Ascham, Platz, 
and Steenbcck '(77), Wilson and Roy (lO), Gytfrgy (28), Dimick and Schreffler (15), 
Halliday and Evans (3^), Edgar, El Sadr, and Macrae (l8). Some of these are 
based on the cure of acrodynia or upon a combination of growth and relief of 
symptoms in rats. The essential fatty acids are concerned in the syndrome, (3, 76) 
together •with pyridoxine since complete cure is not affected by pyridoxine alone. 
The diets were in most cases incomplete since suboptimum growth was obtained even 
with the administration of the vitamin B^ concentrate or crystalline material. 
Further work has since demonstrated that the rations previously used were low in 
a number of factors, the outstanding of which was pantothenic acid. The method of 
Dimick and Schreffler as used loy McElroy and Goss (6k) contained a concentrate, of 
"factor II" but in insufficient amounts. 

The use of chicks for the biological assay of pyridoxine is suggested by the - 
experiments of Heg3ted et al (kO) in which the essential nature of the vitamin 
for chicks was demonstrated. Recent communications by Hogan and his associates 
(U5) and Carter and O'Brien (8, 9) have described rations which can be used for 
qualitative determinations of the vitamin. No published rations so far have been 
complete enough to place the assay on a more quantitative basis. Experiments 
with dogs designed to test for pyridoxin are not advisable for several- reasons. 
There are a number of factors still to be clearly defined in the nutrition of the 
dog which would make it necessary to include some natural material free from 
vitamin Bg. Although such a preparation could be made, the time element is also 
a factor in the assay with this animal. 

Biological Rat Assay Method for Estimating Pyridoxine 

The synthetic pyridoxine low diet that was used in the assays is essentially 
that of Conger and Elvehjem (13) and has the following composition. 



Labco casein 
Sucrose 
Salts 
Corn oil 
Thiamin 
Riboflavin 
Nicotinic Acid 
Choline 

Calcium pantothenate 
Fullers earth filtrate 
of liver powder 1:20 
Halibut liver oil 



18 

75 
k 

3 

200 mcg./lOO gm, of ration 

POQ t! tl tt It tt 

2 
30 

1 



5 mg./lOO gm. of ration 
mg./lOO gm. of ration 
mg./lOO gm. of ration 



If 

1 drop twice weekly 



The liver preparation included in this ration at a k per cent level is low 
in pyridoxine. After attempts to use a variety of liver fractions such as a 
direct butanol extract on the liver powder, an acid ether extract, it was found 
that the fullers earth treated butanol extract of liver extract powder was the 
most satisfactory for our purposes. Although the fullers earth adsorption re- 
moved riboflavin and- thiamin, these crystalline substances were added to the 
basal ration. Other crystalline factors were also added so that adequate amounts 
of these substances would be supplied. 



The fullers earth liver filtrate was prepared by using liver powder, 1:20, 
which is a pernicious anemia fraction of pork liver. Although the primary pur- 






156 

pose of this liver fraction, was not to supply the vitamin B complex, it did 
serve as a good source of most of the water soluble factors. One hundred grams 
of the powder was dissolved in 100 ml. of distilled water and shaken with 500 .ml. 
of "butanol in a mechanical shaker for 2 hours. The "butanol water mixture was 
allowed to settle and the butanol layer removed bj decantation. Five hundred 
ml. of additional butanol were added and the mixture again shaken for two hours. 
The butanol layer was again decanted. This procedure was repeated for five 
times in all. When* anhydrous butanol was used it was found that a small amount 
of water was necessary to aid solution of the liver after the third shaking so 
that more adequate extraction could be obtained with the butanol. The combined 
extracts were concentrated under reduced pressure to a syrup and 200 ml. of water 
was added and the mixture again concentrated in vacuo to remove any traces of 
butanol which were present. The residue after distillation was taken up in 100 
ml. of water and the acidity adjusted to pH 1 to 2 with concentrated hydrochloric 
acid. Five grains of English fullers earth were then added and shaken for 20 
minutes before filtering through a thin pad of washed asbestos (filter eel). Tho 
fullers earth pad was washed with 10-15 ml. portions of N/l0 HC1. This adsorption 
process was repeated four more times using 5 g m . of fullers earth each followed 
by the acid wash. The combined fullers earth filtrates were neutralized to pH 7. 
with NaOH and concentrated under reduced pressure. The concentrate was then made 
up to a convenient volume with the least amount of water. 

In order to prepare the ration, it was found expedient to dry the liver con- 
centrate on the purified casein together with the solutions of the crystalline 
vitamins, The material was placed in a current of warm air' which usually dried 
the liver fraction and the solutions of the vitamins within 12 hours. The salts 
and sucrose were then added and the entire ration was finely ground. The corn 
oil required in the ration was added to 200 gram lots of the ration. These small 
quantities of ration sufficed for 4-5 days 1 feeding and were kept in the refriger- 
ator. In this way there was less opportunity for the ration to become rancid. 

In each bioassay series 60 to 75 rats were obtained at the same time from a 
commercial rattery. These rats were twenty-one day old .male albino rats weighing 
35-I1-O gm. They were placed in individual cages and fed the basal ration for a 
two -weeks depletion period. During this period an average gain of 10 gm. the 
first week and 7 gm. the second week was obtained. After this preliminary period 
in which the attempt was made to equalize the rats before being placed on experi- 
ment, part of the animals were given various meat supplements and the remainder 
were given the various' levels 'of crystalline pyridoxine to furnish the standard 
response for purposes of calculation of the actual pyridoxine content. The con- 
trol rats which were continued on the basal for the experimental period gained 
an average of 5 gm.. per week for the five-week period. The standard pyridoxine 
was included in the rations at levels of 50, 75, and 100 micrograms per 100 gm. 
of ration. The meats that were fed were included in the ration at two or more 
levels and fed to groups of at least two rats, and at times as many as six rats 
were used for each level of meat. 

The rats were examined daily and weighed at weekly intervals. Wo acrodynia 
or other gross symptoms were observed during the five weeks assay period. At the 
end of the assay period the vitamin Bg content of the meats was calculated by 
comparing the growth of the rats on the meat supplement with the growth of the 
rats receiving crystalline pyridoxine (vitamin Bg hydrochloride). After a number 
of preliminary experiments, it was apparent that calculations made on the basis 
of the percentage meat in the ration were more consistent than when the calcula- 
tions were made with the aid of food consumption records. 



157 ■ 

The vita m in Bg hydrochloride content of the meats was calculated by using 
the curve of the growth response of the rats to the crystalline vitamin. The 
average weekly growth of the rats in grams on 50, 75, and 100 micrograms of 
pyridoxine was plotted on a chart. The gain in weight of the rats receiving a 
meat supplement was so controlled that the level of meat used would produce growth 
responses that were in the range of the straight line portion of the calibration 
curve. Table XVIII lists the pyridoxine (vitamin Bg hydrochloride) content of 
the tissues assayed in terms of micrograms per gram of fresh or dried tissue. The 
muscular tissues are very good sources of vitamin Bg. The range of most of the 
muscles from- beef , veal, pork and lamb is approximately from 3.0 to 7*0 micro- 
grams ver gram of fresh tissue. The kidneys of the various species show an 
appreciable pyridoxine content equal to that of the more potent muscle tissues. ; ' 
The livers are next in potency and contain about 3 to k micrograms of pyridoxine 
per gram of fresh tissue. Heart is a moderately good source of the vitamin. 
Beef pancreas contains more of the vitamin than beef lung, beef spleen and beef 
tongue. The cod and salmon samples which were assayed were good sources of 
vitamin Bg and compare very favorably with the high potency ham samples. 

In fluence of Processing on the Pyridoxine Content of Meat 

It seemed worthwhile -to determine the percentage loss of vitamin Bg due to 
commercial processing and by the household methods of cooking. The boiled ham 
samples showed approximately a loss of 50 per cent from the value of untreated 
ham samples.. The tenderized ham samples showed a loss of approximately 60 per 

cent. 

The ordinary household method of frying retained more than two -thirds of its : 
original potency, but the stewed sample showed a loss of about 35 per cent. The 
roasted samples of pork contained about half their original potency. The fried 
pork sample showed about 30 V eT cent loss, whereas the fried beef round showed 
only a slight loss. 

Discussion of Pyridoxine Assays '" 

It is difficult and of questionable value to compare the figures we obtained 
for the pyridoxine content of meats with the few values reported for the "anti- 
acrodynia" potency of meat3 reported by Gyorgy (28), and Schneider, Ascham, Platz 
and Steenbock (77). The only report of the vitamin Bg content of meats by the use 
of a chemical method was presented by Swaminathan (89). He gave 13.^ 'meg. per 
gm. for fresh sheep liver and ^.5 meg. per gm. for, sheep muscle.'. His value' for' 
lamb liver is considerably higher than ours while the values for lamb muscle are 
in the same range. In a sample of milk which we assayed by our animal method we 
obtained 1.3 meg. per ml. of milk, which compares very favorably with the value 
of 1.7 which Swaminathan obtained by his chemical method. ; • i 



The assays reported here indicate that muscle' tissue must be considered a 
better source of vitamin Bg than liver. It should be emphasized that considerar 
ble variation in the vitamin Bg content of a given tissue may be encountered if 
several samples are examined. However, the assay of a few samples of each type 
of tissue gives values which allow a comparison of one meat with another and with 
other foodstuffs. Kidney also is a good source of the vitamin and compares very 
favorably with muscle meats. In all previous assays for the vitamin content of 
meat, kidney and liver were nearly equal in their potency of a particular vita- 
min, but it appears that the vita.min Bg potency of kidney is superior to that of 
the liver of the various species-. Heart is again intermediary in pyridoxine con- 
n- 



tent, being higher than brain, 



Lung, pancreas, and spleen, but lower than liver 



158 

and the muscular tissue. A comparison of some of the available data on the 
vitamin B5 content of foodstuffs wa3 given by Waisman and Elvehjem (97). 

In a previous publication by Henderson, Waisman and Elvehjem (4l) it was 
postulated that the human requirement for pyridoxine was very similar to that of 
thiamin and riboflavin. Such an estimation is acceptable if the requirements of 
the rat can be used as a rough approximation in getting at the requirement of 
other species. It was intimated that the requirement was about 2 .milligrams per 
day for the normal human adult. Using this figure it can be calculated that 
meat furnishes a large share of the daily human requirement of this vitamin. 
Since the average vitamin Bg content of muscle tissue is about 5 to 6 micrograms 
per gram of the fresh meat, and taking into consideration the los3 in cooking, 
approximately 1+00 to 500 grams of muscle meat would be necessary to furnish all 
of the daily requirement. 

TABLE XVIII 
Pyridoxine Content of Meats 



Sample 



Sample 
Number 



Vitamin Bg Hydrochloride 
fresh dry 



mc 



g./gni. 



mcg./gm. 



Beef 


kidney 


126 


ti 


ti 




81 


11 


11 


- (Stewed) 


112 


Pork 


kidney 


83 


it 


it 




137 


Pork 


ham 


(dried) 


95 


it 


ti 


tt 


124 


it 


it 


tt 


139 


it 


it 


(fresh) 


i4o 


ti 


tt 


(fried) 


l4l 


<r 


it 


(roast) 


l42 


it 


t! 


(fried) 


52 


tt 


tt 


(boiled) 


115 


!t 


II 


(Smoked) 


116 


It 


tt 


(tenderized) 


72 


It 


It 


tt 


119 


It 


It 


tt 


117 


Pork 


loin 


. 


125 


ti 


11 




89 


tt 


tt 




■' 36 


11 


tt 


(roasted) 


94 



4.3 
4>4 

4.0 
7.0 
6.0 
6.7 
5.5 
5.9 



2.9 

2.4 
2.9 
3.8 
5.1 
6,5 
4.5 



21.3 

19.5 
14.4 
18.1 
34.8 
24.6 
28.2 
21.7 
23.5 
15.7 
12.1 
17.6 
10.6 
9.8 

7.7 
8.8 

11.0 

18.8 

19.5 
14.5 
11.5 



TABLE XVIII (Continued) 



159 





Sample 


Saraple 
Number 


Beef 


muscle 


128 


tt 


it 


105 


it 


" (fried) 


106 


Lamb 


muscle 


80 


Veal 


muscle 


129 


it 


it 


13* 


it 


n 


103 


Beef 


liver 


131 


Lamb 


liver 


96 


Pork 


liver 


86 


it 


it 


122 


Veal 


liver 


97 


Beef 


heart 


73 


Pork 


heart 


104 


Beef 


"brain 


77 


Beef 


lung 


138 


Beef 


pancreas 


115 


Beef 


spleen 


13C 


Beef 


tongue 


82 


Chicken muscle (dark) 


66 


Frozen fillet of cod 


135 


Frozen salmon steak 


136 



Vitamin Bg Hydrochloride 
fresh dry 



;g./gm. 


mcg./gm. 


3.8 


16.2 


4.0 


14.5 


-- 


12.0 


3.0 


11.8 


4.0 


16.2 


4.2 


19.4 


4.4 


16.3 


7.3 


25.0 


3.7 


12.6 


3.2 


9.9 


3.3 


10.3 


3.0 


10.3 


2.4 


11.2 


3.5 


15.6 


-- 


5.3 


0.7 


3.5 


2.0 


7.5 


1.2 


5.7 


1.2 


4.2 


2.0 


8.0 


3A 


19.6 


5.9 


21.2 



L 



161 



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flavinosis. U. S. Public Health Reports, 5V, 790 (1939). 



165 



70. Physiological Activity and Experimental Clinical Use of Vitamin Bg. Merck 
and Co. Annotated Brochure, Sept. ( 194-0 ). - 

71. Quackenbush, F.W., and Steenbock, H. Acrodynia and the Essential Fatty Acids* 
J. Biol. Chem., 125 xovii (1938). 

72. Richter, C.P., Holt, L.E., Barelare, Jr., B. and Hawks, CD. Changes in Fat, 
Carbohydrate, and Protein Appetite in Vitamin B Deficiency. Am. J. Physiol., 
124, 596 (1938). 

73. Bobbins, W.J. and Schmidt, M.B. Vitamin Bg as a Growth Substance for Excised 
Tomato Roots. Proc. Natl. Acad. Sci., 2^ 1 (1939). 

74. Salmon, W.D. The Effect of Certain Oils in Alleviating Localized Erythematous 
Dermatitis (Acrodynia or Vitamin Bg Deficiency) in Rats. J. Biol. Chem., 

123, oiv (1938). ';' 

75* Salmon, W.D. The Supplementary Relationship of Vitamin Bg and Unsaturated 
Fatty Acids. J. Biol. Chem., 133, lxxxiii (I9k0) . 

76. Schneider, H.A., Steenbock, H., and Platz, B.R. Essential Fatty Acids, 
Vitamin Bg and Other Factors in the Cure of Rat Acrodynia. J. Biol. Chem., 
132, 539 0-940). 

77. Schneider, H.A., Ascham, J.K., Platz, B.R., and Steenbock, H. The Anti- . . 
acrodynia Properties of Certain Foods. J. ITutr., l8, 99 (1939). 

78. Schultz, A.S., Atkin, L., and Frey, C.N. Vitamin Eg, a. Growth Promoting 
Factor for Yeast. J. Am. Chem. Soc, 61, 1931 (1939). ' 

79. Scudi, J. V., Bastedo, W.A., and. Webb, T.J. The Formation' of a Vitamin Bg- 
Borate Complex. J. Biol. Chem., 136, 399 (1940). 

80. Scudi, J. V., Koone3, H.F., and Eeresztesy, J.C. A Colorimetric Method for 
the Determination of Vitamin Bg. Am. J. Physiol., 129, V?9 (19I+O). 

3l. Scudi, J. V., Kb one s, H.F., and Eeresztesy, J.C. Urinary Excretion of 
Vitamin Bg in the Rat. Proc. Soc. Exp. Biol, and Med., _43, 118 (1940). 

82. Sebreli, W.H., and Butler, R.E. Riboflavin Deficiency in Man. A Preliminary 
Note. U. S. Public Health Report, J£L 2282 (1958). 

83. Smith, S.G., and Martin, D.W. Cheilosis Successfully Treated with Synthetic 
Vitamin Bg. Proc. Soc. Exper. Biol. Med., j£^ 660. (1940). 

84. Spies, T.D., Bean, W.3., and Ashe, W.F. A Note on the Use of Vitamin-Bg 
in Human Nutrition. J. Am. Med. Assoc, 112 . 2412 (1940). 

35. Spies, T.D., Eightower, D.P., and- Hubbard, L.H. Some Recent Advances in 
Vitamin Therapy. J. Am. Med. Assoc, 115. 292 (1940). 

86. Spies, T.D., Ladisch, R.K., and Bean, W.B. Vitamin Bg (pyridoxine) Deficiency 
in Human Being3, J. Am. Med. Assoc, 115. 839 (1940). 

87. Stiller, E.T., Eeresztesy, J.C, and Stevens, J.R. The Structure of Vitamin 
Bg. J. Am. Chem. Soc, 61, 1237 (1939). 



166 

88. Sullivan, M. , and Nicholls, J. The Nutritional Approach to Experimental 

,-_ Dermatology. Nutritional Dermatoses in the Eat I. Vitamin B<5 Deficiency. 
J. Invest. DermJ>, 317 (19^0), 

89. Swaminathan, M. Chemical Estimation of Vitamin Bg in Foods "by Means of the 
Diazo Reaction and the Phenol Reagent. Nature 1^5, 7o0 (19^-0). 

• 

90. Svaminathan, M. A Chemical Test for Vitamin Bg in Foods. Ind. J. Med. 
Res., 28, 42? (19^0). • ■ 

91. Tange, U. Studies on Vitamin Bo Complex VI. Rat Acrodynia and Fatty Acids. 
Sci. Papers Inst. Phys. Chem. Res., J>6, J +82 ( 1939 ^ . : 

92. Unna, K. Toxicity and Pharmacology of Vitamin B5. Am. J. Physiol.. 129 , 
U83 (19^0). 

93- Unna, K. Pharmacology and Toxicology of Vitamin Bg (as quoted in Merck 

Brochure on Experimental and Clinical Use of Vitamin Bg) J. Pharm. and Exp. 
Ther . (in press) . 

9^. Unna, K., and Antopol, W.- Toxicity of Vitami^ B5. Proc. Soc. Exp. Biol. 
and Med., j£, ll6 (19I+O). ■ ■ - 

95. Vilter, R.W., Schiro, H.S., and Spies, T.D. Effect of Synthetic Vitamin B5 
on the Hemopoeitic System of Human Beings. Nature, 14*3, 388 (l9'+0). 

96. Vilter, S.P., and Spies, T.D. Vitamin Bg as an Accessory Growth Factor for 
Staphylococcus Alhus . Science _91, 200 (19^0). 

97. Waisman, E.A., and Elvehjem, C.A. Chemical Estimation of Nicotinic Acid 
and Vitamin Bg . Ind. Eng. Chem., Anal. Ed., 13, 221 (19^1) 

98. Waisman, H.A., and Elvehjem, C.A. Multiple Deficiencies of the Goldherger 
Diet as Demonstrated with Chicks. J. Nutr., 20, 519 (19^-0). 

99- Waisman, H.A ; ., Henderson, L.M. and Elvehjem, C.A. (Unpublished data) 19^1. 

100. Wegner, M.I., Booth, A.N., Elvehjem, C.A. and Hart, E.B. Rumen Synthesis 
of the Vitamin B Complex. Proc. Soc. Exp. Biol, and Med., k5, 1&9 (19^0). 

101. Wilson, H.E.C. and Roy, G.K., Flavine and Vitamin B< (Antidermatitis Con- 
tent) of Indian Foodstuffs. Ind. J. Med. Res., 2^, 879 (1938). 

102.. Woolley, D.W. and Hutchings, B.L. Synthetic Media for Culture of Certain 
Hemolytic Streptococci J. Bact., 39, 287 (19^0). 

103. Zelig's, M.A. Use of Pyridoxine Hydrochloride' (Vitamin B/0 in Parkinsonism. 
J. Am. Med. Assoc.; 116, 2l48 (19^1). 



CHAPTER XIII 

PANTOTHENIC ACID 

Page 
History and Development of the Factor 167 

Identification of Pantothenic Acid as the Chick 

Anti dermatitis Factor l68 

Chemistry of Pantothenic Acid l68 

Role of Pantothenic Acid in _>trition 169 

Bioassay for Pantothenic Acid with Chicks 171 

Effect of Household Cooking Processes on Stability 

of Pantotheni c Ac i d 177 

Discussion on the Chick Assays 177 

Microbiological Assay for Pantothenic Acid 178 

Metho d of Assay 178 

Results of Microbiological Assay l8l 

Discussion of Pantothenic Acid Results 185 

Literature Cited ... . 187 



L 






CHAPTER XIII 

PANTOTHENIC ACID 

The increasing significance of pantothenic acid in the normal nutrition of 
many species of animals has demanded that we give greater attention to the 
occurrence of this vitamin in many of our natural foodstuffs. Since the avail- 
ability of the crystalline synthetic vitamin to many laboratories, the importance 
of this substance for normal growth and for normal metabolism has been amply 
demonstrated. It has been shown to be required by the chick, rat, .mouse, dog 
and pigeon, and only within recent months has it received attention by the 
clinician in its application to some dietary deficiencies in .man. 

History and Development o f t he Fac tor 

The original symptoms of chick dermatitis were described by KLine, Keenan, 
Elvehjem. and Hart (26), Morris and Ringrose (kk) and Ringrose, Norris and Heuser 
(52). The typical incrustations at the corners of the beak, scaliness of the 
legs and sticky eyelids was most simply obtained by dry heating a natural grain 
ration consisting of yellow corn and wheat middlings.. Depending upon the origi- 
nal dietary history of the birds, the syndrome was of increasing severity as 
indicated by the condition of the feathers and the skin areas of the eyes, legs 
and mouth. The apparent similarity of these symptoms to human pellagra was the 
basis for referring to the chick condition as "chick pellagra". Shortly after 
the description of the typical chick symptoms by the Wisconsin and Cornell groups, 
Lease and Parsons (30) claimed that the symptoms observed in chicks fed unheated 
egg white were identical with those found in chicks fed a ration low in the 
dermatitis factor of Kline and coworkers and of Norris et al. Lease and Parsons 
concluded however that the "egg white injury factor" and the chick anti dermatitis 
factor were separate entities. 

Several investigators at this time became interested in the isolation of the 
active material in liver which would prevent the dermatitis symptoms in the chick. 
The name "vitamin B2 or G" was given earlier to the heat stable factor of the B 
group of vitamins thus differentiating it from vitamin Bj . It soon became appar- 
ent from the researches of several groups of workers that more than one substance 
was concerned in the vitamin Bg factor. The substance which would cure the 
typical "rat pellagra" symptoms could be absorbed on fullers earth while the 
active concentrate which would prevent the dermatitis in the chick was made from 
the filtrate of the fullers earth adsorption. Early work had also furnished 
evidence that a pigment was also absorbed and it was subsequently shown that the 
pigment was riboflavin. When it was found that riboflavin was part of the 
vitamin B2 or G factor, the yellow pigment was fed to chick3 maintained on a 
dermatitis producing ration, but the .material was found to be inactive in pre- 
venting the syndrome (.10). The administration of riboflavin to black tongue . 
dog3 resulted in the finding that this vitamin was inactive in the cure, or pre- 
vention of the typical "pellagra symptoms". Lepkovsky and Jukes (31) confirmed 
the finding of Slvehjem and Koehn (10) and in addition concluded that the. 
"aqueous extract of liver contains two factors", riboflavin and another factor, 
both of which were distinct from vitamin B-i . It was. shown in later work by 
Lepkovsky and Juke3 (32) that the treatment of an aqueous extract of liver with 
fullers earth would separate the factors of the vitamin B2 complex. Lepkovsky, 
Jukes and Erause (33) elaborated on their term "filtrate" factor", and attempted 
to establish the existence of this third factor of the B'2 complex. One part of 
tMa third factor cured dermatitis in rat3 but had no effect on the chick derma- 
titis. Conversely the other portion of the third factor cured or prevented the 
dermatitis in the chicks but had no effect on the rat dermatitis. This latter 

-I.67- 



168 

fraction was the filtrate of the fuller a earth adsorption and the California 
workers thus called it the "filtrate factor". The growth response to suboptimal 
levels of the "filtrate factor" was roughly proportional to the amount fed 
according to the work of Jukes and Lepkovsky (33). Further work by Jukes (23) 
pointed out the dissimilarity which existed in the distribution of the factors 
which prevented the typical symptoms in dogs and the dermatitis symptoms in 
chicks. The discovery (12) that nicotinic acid wa3 the anti-black tongue factor 
allowed a test of the hypothesis that the condition of black tongue in dog3 and 
dermatitis in chicks resulted from the same deficiency. It was shown by us, 
Mickelsen, Waisman, and Elvehjem (Ul) and also by Dann and Subbarow (5) that 
nicotinic acid was entirely inactive in the cure or prevention of the dermatitis 
in chicks fed the heated grain diet. 

Identification of Pantothenic Acid as the Chick Antidermatiti s Factor 

The fractionation of liver for the chick antidermatitis factor in this 
laboratory resulted in the finding that the active principle was an acidic com- 
pound and extractable from acidified liver by prolonged ether extraction. The 
paper by Woolley, Waisman, Mickelsen, and Elvehjem (68) recorded the early ob- 
servations made on the chick antidermatitis factor. Further study resulted in 
the fractionation of active concentrates to yield highly potent material. The 
nature and partial synthesis of the factor was soon after announced by Woolley, 
Waisman and Elvehjem (69, TO). These workers showed that the chick antidermatitis 
was very similar in properties to pantothenic acid, and that the factor was a 
hydroxy acid in amide combination with ]6 alanine. A similar finding was recorded 
by Juices (2^). These were the first indications that pantothenic acid had some 
part in animal nutrition. Williams (60) commented on the discovery by the Wiscon- 
sin and California workers and welcomed the "confirmation of a suspicion which 
has long been entertained -~ namely that pantothenic acid is a vitamin of import- 
ance in animal nutrition." 

Chemistry of Panto thenic Acid 

Pantothenic acid is chemically defined as oc, if, dihydr oxy-/3,/3>- dimethyl - 
butyryl-/3' -alanide. It has the structure: 

— C~ 0-H 



H CH X OH 


>> 

-C 


H 
-If— 


H 
— C 


I H 






rr 


CHj 


'. 







H ! H H H 



The compound was first called "pantothenic acid" ~by Williams and coworkers 
(67). It was believed to be a growth determinant of universal biological 
occurrence. Since the first publication in 1933, Williams and his associates 
have contributed many studies on the properties and the possible structure of the 
compound. The availability of highly active concentrates of the vitamin enabled 
the characterization of the material. Following the partial synthesis of the 
vitamin described by Woolley, Waisman and Elvehjem (69), the isolation and • 
structure of the lactone moiety was reported by Stiller, Keresztesy and Finkel- 
stein (58). This was followed by a partial and t^tal synthesis' by Williams and 
his associates (65). The details, of the total synthesis. of pure pantothenic 
acid ha3 been reported by Stiller, Harris, Finkelstein, Keresztesy and Folkers 
(57). The synthetic preparation of pantothenic acid now available commercially 
is the dextrorotatory- calcium pantothenate. 



L 



169 

Pantothenic acid is a very water soluble compound. Most- of its salts are. 
also very soluble in water. The calcium and "barium salts are soluble in 95 V er 
cent alcohol but insoluble in absolute ethyl alcohol. The substance is readily 
hydrolyzed into its two component parts, especially in the presence of small 
amounts of alkali. It is also lieat labile and is lactonized readily by dry 
heat. Autoclaving pantothenic acid or its salts in both acid and alkaline solu- 
tions will also destroy its vitamin properties. No melting point is available 
at present of the pure compound, since it has been difficult to prepare the free 
acid in crystalline form. The optical rotation of the dextrorotatory calcium 
salt of pantothenic acid is [a] §^= + 2^.93°. 

A number of related compounds have been tested for their biological activity, 
Mitchell and coworkers (^2) have shown that hydroxy pantothenic acid, the hydroxyl 
group replacing one of the hydrogens from one of the /3 methyl groups, has varying 
activity depending upon the microorganism which is used for the test. These 
workers previously found that related compounds prepared from ornithine and 
lysine coupled with £ -alanine showed very little activity. Reichstein and 
Grussner ( 50) have also discussed the biological activity of compounds structural- 
ly related to pantothenic acid. It is to be expected that further work on this 
aspect of the problem will demonstrate the chemical groups which are essential 
for the biological activity of pantothenic acid. 

Role of Pantothenic Acid in Nutrition . . 

The first experiments which indicated that pantothenic acid had some part in 
animal nutrition were done in this laboratory (69, 70) and by Jukes (2k) in 
California. It was conclusively shown by these two groups of workers that the 
"universal compount -pantothenic acid" was the chick antidermatitis factor. Panto- 
thenic acid appears to be necessary for reproduction in the domestic fowl as 
shown by the work of Bauernfeind and Norris (l). A histopathological study of 
chicks deficient in the antidermatitis vitamin demonstrated that pantothenic acid 
wa3 necessary for the maintenance of the intact spinal cord (^+8). The lesions in 
the spinal cord were "characterized by the myelin degeneration of myelinated 
fibers distributed widely throughout the white matter, except in the posterior 
regions". 

A hint that rats require pantothenic acid was given first by Edgar and 
Macrae (7) when they differentiated "between vitamin Bg and the "filtrate factor". 
These workers were unable to decide which of their factors was pantothenic acid 
(9) but in a recent paper (3*0 this group identified the liver filtrate factor 
required by their rats with pantothenic acid. Subbarow and Hitchings (-59) showed 
that definite growth responses were obtained in rats fed pantothenic acid. 
Hitchings and Subbarow (l8) gave further evidence for the pantothenic acid acti- 
vity as part of the liver fraction which promoted growth. Oleson, Woolley and 
Elvehjem (^5) found that /3 -alanine would not replace pantothenic acid as part of 
the growth promoting fraction of liver. Hoffer and Reichstein (19) however, re- 
ported that /•? -alanine would replace pantothenic acid "since rats react in a simi- 
lar way to free /3-alanine and to all ordinary ester and acyl derivatives of 

/^-alanine". Gyc'rgy, Poling and Subbarow (17), Black, Frost and Elvehjem (2) and 
Woolley (72) gave further evidence for the pantothenic acid requirement of the 



Preliminary evidence by McKibbin, Madden, Black and Elvehjem (39) indicated 
that pantothenic acid might be needed by the dog. A further indication of this 
was 3hown in the work of Fouts, Helmer, and Lepkovsky (15) in which a synthetic 
diet was fed to the dogs. The diet was deficient "apparently only in a factor or 
factors contained in a purified liver extract other than nicotinic acid". From 



170 

some observations in this laboratory b,y McKIb'bin et al (^0) it -would appear that 
other factors in the liver extract such as choline and an alkali labile factor 
are also present in the liver extract. 

The role of pantothenic acid in the nutrition of other animals is not ' 
altogether clearly defined. Damashek and Meyerson (h) observed a "pigeon derma- 
titis" which responded to a liver fraction since shown to be a rich concentrate 
of pantothenic acid (70). Hughes (20, 2l) fed pigs a diet deficient in the 
"filtrate factor" and observed that these animals showed an unthrifty appearance 
with paralysis of the legs. The administration of a concentrate containing the 
factor caused an immediate increase in appetitie and physical well being. Sheep 
evidently do not require pantothenic acid in their diet since it is known to be 
synthesized in the rumen of these animals (36). Cattle also are able to synthe- 
size this vitamin in the rumen (37, Sk) t 

The large amount of effort expended on the problem of nutritional require- 
ment of bacteria has established the importance of pantothenic acid in the normal 
growth of a variety of organisms. Williams and his coworkers (67) first demon- 
strated the action of this factor in stimulating the growth of a particular strain 
of yeast. Pantothenic acid was believed to be synthesized by Aspergillus niger 
and confirmation of this was obtained by Gorcica, Peterson and Steenbock (16) who 
found that the addition of as little as one per cent of the dried mycelium of 
Aspergillus sydowi to a ration low in pantothenic acid was sufficient to prevent 
the typical dermatitis in chick3. Elliott (8) found that pantothenic acid stimu- 
lated the growth of some protozoa but not others. Richards (51) reported the 
stimulation of yeast proliferation by pantothenic acid which verified much of the 
work of Williams up to that time. A possible mechanism for the action of panto- 
thenic acid in certain microorganisms has been offered by McBurney, Bollen and 
Williams (35) when they described the production of pantothenic acid by Rhizobium 
meliloti which is one of the symbiotic bacteria important in the nitrogen fixation 
process of legumes. 

Snell, Strong, and Peterson (55, 56) asserted that their factor necessary 
for Lactobacillus casei e was identical with pantothenic acid, and Koser, Finkle, 
Dorfman, Gordon and Saunders (29) also showed some activity of pantothenic acid 
for their strain of diphtheria bacillus. The use of [b -alanine for growth re- 
sponses in certain organisms was resorted to in certain investigations on the 
growth requirements of a variety of organisms. Mueller, and KLotz (kj) could not 
show any stimulation to £> -alanine but could with pantothenic acid. When a 
hydrolysate of the growth concentrate was added, there was slow growth, and the 
authors concluded that "/^-alanine must first -be built up into some more complex 
material before it may be directly utilized.." Mcllwain (38)' and Woolley and 
Hutchings (71) foimd that very small amounts of pantothenic acid were required 
to stimulate the growth of Streptococcus hemolyt icus. Investigations by Evans 
and his associates (13) on Corynebacterium diphtheriae , Pelczar and Porter (46) 
on Morgan's bacillus and Subbarow and Sane (59) on a particular strain of 
Hemolytic 3treptococcu3 , gave final proof that these organisms required panto- 
thenic acid for their normal growth. 

An interesting observation has been made with the growth of pea embr5 r os in 
v itro . Pantothenic acid together with other members of the B group of vitamins 
caused a marked stimulation in growth of the plant embryos (3). This "hormonal" 
type of stimulation has also been reported by Thimann and Bonner (6l) and by 
Thomas (62). 



171 



Bioassay for Pantothenic Acid with Chick s 



The assays for pantothenic acid in meats were undertaken in an effort to get 
a relative comparison of the potency of the various animal tissues. Although 
preli m inary experiments were made to get the approximate range of levels of the 
meat to "be used for the -assays, later experiments were designed so as to compare 
the minimum protective level of the meat with the minimum amount of pantothenic 
acid that would just give protection against the dermatitis. It was felt that 
such a procedure would he a more reliable indication than growth, since some 
work "by the authors (63) had shown that the heated diet used for the assays was 
primarily deficient in pantothenic acid "but "borderline in a number of other 
factors. Bauernfeind and Norris (l) have since published on another factor re- 
quired on this particular diet. The minimum level procedure has been received 
with commendation by a number of investigators. Dimick and Lepp (6) have used 
this experimental procedure in their parallel feeding trials of pantothenic acid 
and an amount of rice bran extract that contained an equivalent amount of panto- 
thenic acid. 

The basal ration 2UlH used throughout these assays consists of ground 
yellow corn, 58; wheat middlings, 25; crude casein, 12; CaCO* 1; Ca,(PO^) 
1; NaCl 1; hexane extract of alfalfa leaf meal or synthetic 2 -methyl, 1, k 
naphthoquinone. The corn, middlings and casein were heated for 30 hours at 120°C, 
in a thermostatically controlled oven. The hexane extract of alfalfa or the 
naphthoquinone supplied sufficient vitamin K. Vitamins A and D were furnished by 
twice weekly drops of halibut liver oil. The basal diet was supplemented with 
100 micrograms of thiamin and 200 micrograms of riboflavin per' 100 grams of 
ration. In later experiments pyridoxine was also added to the basal ration, but 
in no case was there any effect of the added vitamins on growth or on the pre- 
vention of the symptoms. The addition of choline also had no effect on the 
dermatitis or upon growth. 

The White Leghorn chicks which we used for our assays were obtained from 
commercial hatcheries and from the poultry department of the University of 
"Wisconsin, The baby chicks were placed in heated brooders on raised screens in 
groups of 6 and were observed daily for dermatitic symptoms on the legs, eyes, 
and beak. The assay period was usually for 5 week3, during which time weekly 
notations were made on the gain in weight. In all cases the minimum level of 
the meat required to give complete protection was determined by feeding a number 
of levels of the meat. The percentage of meat fed on the basal diet usually 
included levels higher and lower than the minimum protective level. The lowest 
level that furnished protection from symptoms was finally arrived at by harrowing 
down the levela on either side of the minimum. It was found that the levels of 
the tissue higher than the minimal protective level showed corresponding graded 
increases in growth and decreased incidence of symptoms. Typical weight incre- 
ments in growth and protection from the symptoms with various levels of meat are 
presented in table XIX. The graded increases in weight were also accompanied by 
increased protection from dermatitis. Many of the series were run for 6 weeks 
although similar conclusions can be drawn from the results after 5 weeks. 



In table XX complete data on the chick assays are given. The minimum pro- 
tective level for a particular tissue is summarized at the end of the group of 
similar tissues. Liver is one of the outstanding sources of any of the tissues 
tested. Teal, beef, and lamb liver furnish complete protection to the chicks at 
the same level. Although increasing weights of the birds resulted from the 
increasing levels of the meat, it is interesting to note that nearly the same 
average weight was obtained with 2 per cent of any of the livers. It is import- 
ant to direct attention to the fact that although a borderline protection was 
furnished by 2 per cent of the pork liver, the average weight of the group was 
more than twice that of the basal control group. 



172 



TABLE XIX 
Typical Increments in Growth of Chicks Fed Increasing Levels Of Tissue 





Sample 


Level 


Ave . wt . 


No. of 


No. with 


No. Dead 


Sample 


No. 


fed 


5 wks. 


chicks 


symptoms 


5 wk3. 


Beef heart 


73 


3 


139 


k 


.3 







73 


5 


162 


h 










53 


10 


223 


k 





1 




13 


10 


225 


k 










13 


20 


228 


h 










53 


20 


276 


k 








Beef lung 


78 


10 


95 


k. 


2 


1 




78 


, 20 


135 


k 


1 







6 


30 


172 


k 


■ • 







6 


ko 


199 


k 


■■ , 





Beef pancreas 


79 


5 


122 


H 


2 


- 1 




79 


7.5 


150 


k 


2 


2 




79 


10 


1^3 


5 


• 







79 


20 


198 


h 








Lamb liver 


61 


2 


163 


6 ', 


o : 







61 


3 


216 


k 










6l ' 


k 


223 


k 








Pork liver 


29 


0.5 


90 


5 


k 


■ 1 


< 


29 


1 


122 . 


k 


3 


0' 




29 


2 


167 


k 


1 





. 


29 


■k -, 


223 


8 








Stewed beef 


87 ; 


5 


125 


9 


.',. 5 


0' 


.. . heart 

■ 


87 


7.5 


205 


V 





1 




87 ■■■":■ 


10 


272 


k 


' .. 









173 



Sample 



TABLE XX 
Pan tothenic A ssays Wit h Chicks 



Approximate 
Pantothenic Acid 
Sample Level Ave. Wt. Min. Prot. Micrograms/Gm 

Number Fed 5 wks . Symptoms Level Dry Wt. 



Beef Brain 



Beef Heart 



Beef Kidney 



Beef Liver 



Beef Lung 



77 


5 


199 


57 


10 


181 


57 


20 


178 


11 


20 


23^ 


11 


30 


183 


73 


3 


139 


73 


5 


162 


73 


10 


2U6 


53 


10 


223 


13 


10 


225 


13 


20 


228 


53 


20 


276 


8U 


1 


158 


Qk 


2 


235 


8>k 


3 


157 


k9 


3 


208 


9 


3 


200 


9 


5 


165 


h9 


5 


212 


8k 


5 


196 


58 


1 


165 


98 


1 


1*7 


10 


2 


191 


58 


2 


.154 


98 


2 


197 


10 


* 


220 


98 


k 


183 


78 


10 


95 


78 


20 


135 



60 



60-100 



150-200 



150-200 



+ 
+ 



174 



TABLE .XX (Continued) 



Sample 



Beef Lung 



Beef Muscle 



Beef Spleen 



Beef Pancreas 



Beef Tongue 



Lamb Kidney 



Lamb Liver 



Approximate 
Pantothenic Acid 
Sample Level Ave. Wt. Min. Prot. Micro grams/ Gm 

Number Fed 5 wks. Symptoms Level Dry Wt. 



. 6 


30 


172 


6 


4o 


199 


19 


20 


157 


18 


20 


145 


40 


20 


127 


105 


25 


170 


19 


30 


142 


ko 


30 


142 


105 


35 


187 


76 


5 


97 


76 


10 


154 


k 


10 


174 


76 


15 


136 


4 


20 


268 


19 


5 


122 


79 


7.5 


150 


79 


10 


143 


79 


20 


198 


7 


20 


ikf 


82 


5 


187 


82 


10 


111 


22 


5 


187 


22 


10 


246 


96 


l 


160 


96 


2 


198 


23 


1 


140 


6l 


2 


163 


6l 


3 


216 


61 


4 


223 


23 


4 


192 


23 


10 


231 



+ 
+ 



+ ' 
+ 



25 



25 



10-15 



10 



15 



12-30 



12-15 



20-35 



30-50 



30-40 



less than 5 J.25-I5O 



250-300 



TABLE XX (Continued) 



175 



Sample 



Sample Level Ave. Wt. 
Number Fed 5 wl-rs . 



Approximate 
Pantothenic Acid 
Min. Prot* Micro grams/dm 
Symptoms Level Dry Wt. 



Lamb Muscle 




80 


25 


188 


+ 






35 


30 


196 


- 


Fork Kidney 




83 


1 


182 


+ 






33 


2 


169 


+ 






62 


2 


115 


- 






28 


3 


168 


- 






62 


k 


223 


- 






28 


5 


166 


- 


Pork Liver 




29 


0.5 


90 


+ 






29 


1 


122 


+ 






86 


1 


9^ 


+ 






86 


2 


159 


+ 






29 


, 2 


167 


+ 






29 


k 


223 


- 


Pork Muscle 


-Ham 


12 


20 


I69 


- 




-Loin 


30 


20 


117 


+ 




tt 


2h 


20 


115 


- 


Shoulder 


25 


20 


129 


+ 




Loin 


3h 


20 


206 


+ 




Ham 


32 


20 


115 


+ 




Ham 


51 


20 


I85 


+ 




Ham 


12 


30 


172 


- 




Loin 


30 


30 


100 


+ 




Loin 


2h 


30 


129 






Ham 


26 


30 


135 


+ 




Ham 


51 


30.. 


149 


- 


Smoked Ham 




hi 


20 


153 


- 






hi 


30 


165 




Yeal Muscle 
(Hindquartf 


ar) 


kk 
75 


25 

25 


211 . 
168 


+ 
+ 



30 



12-15 



125-150 



3-* 



75-100 



25-30 



20 



15-30 



15-25 



176 



TABLE XX (Continued) 



Sample 



Approximate 
Pantothenic Ac id 
Sample Level Ave. Wt. Min. Prot. Micrograma/Gm 

Number Fed 5 wks. Symptoms Level Dry Wt. 



Veal Muscle 
(Eindquarter ) 



kk 35 201 

75 35 166 
16 k0 236 



^5-30 



15-25 



Veal Liver 


97 


1 


66 


+ 




97 


2 


194 


- 




5 


2 


182 


- 




5 


k 


263 


- 


Fried Beef Liver 


99 


1 


132 


- 




99 


2 


238 


- 




99 


I* 


202 


- 


Stewed Beef Heart 


87 


5 


125 


+ 




87 


7.5 


205 


- 




87 


10 


272 


- 


Stewed Beef Spleen 


83 


5 


125 


+ 




83 


10 


115 


+ 




88 


15 


103 


+ 


Stewed Beef Kidney 


85 


3 


122 


+ 


« 


85 


5 


161 


- 



1-2 



7.5 



15 



150-200 



150-300 



30-^0 



17-20 



60-75 



Kidney was as active as liver as a source of the vitamin and "beef heart 
showed surprisingly high amounts of pantothenic acid in contrast to the other 
muscular tissues. Beef spleen was next in potency. Some variation was ob- 
served in the growth response of the beef brain samples, but the minimal pro- 
tective level was the same for all samples of the tissue. Beef pancreas and 
beef tongue were next in order of potency. Beef lung showed the least activity 
of any of the organs . 



The muscular tissue of beef, lamb, pork and veal are fair sources of 
pantothenic acid and are approximately one-tenth as rich as the liyer or kidney. 
The several samples of beef muscle showed some variation in their ability to 
protect against the dermatitis. Similar results were obtained with the lamb 
muscle samples. 



177 



The pantothenic acid content of pork muscle is in the same general range 
as that of beef, veal and lamb muscle. A total of nine samples were assayed 
"by the chick method and these varied slightly in their response. Several of 
the samples produced good growth out did not protect against the dermatitis, 
vhile others protected against the symptoms "but did not show any growth. At 
present, no correlation can he made with- the grade of the carcass, the season 
when the animal was slaughtered, and the minimal protective level. 

Effect of Household Cooking Processes on Stability of Pantothenic Acid 



It waa decided to utilize the higher potency samples for the study of the 
processing procedure on the pantothenic acid content. The liver was fried and 
the heart, spleen and kidney samples were. stewed according to the usual house- 
hold procedure. The graded increase in average weight of the birds fed the 
increasing level of cooked tissue was apparently the same as in the groups fed 
the untreated material. The cooked samples coulc not he compared directly to 
uncooked portions of the same tissue since the latter were not available; 

The sample of fried liver gave fully as good protection as did the untreated 
portion of the same sample. When the untreated sample was fed at a 1 per cent 
level there was incomplete, protection from the typical symptoms, but when the 
fried portion of the same sample was fed at the 1 per cent level no symptoms 
were observed in any of the birds. It is noteworthy that the growth was better 
on the fried sample than on the untreated tissue. This is not due to a fortui- 
tous choice of chicks since the average of two separate series of experiments 
indicated that the 12 birds on the 2 per cent of untreated liver weighed 200 
grams at the end of the five weeks assay as compared to the weight of 2^-1 grams 
for the 10 birds on the fried beef liver. It wa3 also apparent that the 1 per 
cent of fried beef liver protected the birds from dermatitis but did not allow 
as good growth as with 2 per cent. Good growth and ample protection wa,s ob- 
served at the higher level. . . .• 

The ability of the samples of beef heart, beef spleen, and beef kidney to 
furnish protection from the dermatitis is decreased by stewing. The comparative 
growth of the chicks on the cooked and untreated meats again demonstrates a 
lower gain together with a decreased ability to afford protection from the der- 
matitis. It can be concluded that approximately one-third of the potency was 
lost by stewing. This decreased potency of the cooked meats can be best ex- 
plained by the partial extraction of pantothenic acid by the water in which the 
meat was cooked. 



Discussion on the Chick Assays 

The U3e of our. heated basal diet 2^-1 H was fully satisfactory for the esti- 
mation of the relative potencies of pantothenic acid in a large variety of 
mea.t3 . Although these figures are not of a fully quantitative nature, it is 
po33ible to place the values on a more firm foundation by comparing the results 
obtained with the experiments where crystalline pantothenic acid was used (63). 
The level of calcium pantothenate that would just prevent the typical symptoms 
in all the birds in the group was considered to be the minimal pantothenic acid 
requirement. Thi3 must, be differentiated from the requirement of the chick for 
optimum growth.- There can be no doubt that the amount of the vitamin needed for 
prevention' of the dermatitis would be less than that necessary to promote optimum 
growth, and this is borne out "oj the work of Jukes (22), who has set the panto- 
thenic acid requirement of the bird3 at 1^-00 micrograms per 100 grams of diet 
for optimum growth. • 



178 

By knowing the minimal protective level of a particular tissue and knowing 
the minimal amount of crystalline vitamin that will just give protection from 
the dermatitis, the meat can then he considered to contain the .minimal amount of 
the crystalline pantothenic acid. From a long series of. experiments we have 
concluded, Waisman et al (63), that 300 micrograms of crystalline calcium panto- 
thenate per. 100 grams of ration is sufficient to protect the chicks from the 
typical incrustations. Therefore, if the pantothenic acid content of a particu- 
lar liver sample is to he calculated, the level of meat that just furnishes pro- 
tection can he considered to he equal to 300 micrograms. For example, the 
majority of the heef liver samples contained sufficient pantothenic acid so that 
2 per cent was enough to furnish the necessary protection; the 2 per cent was 
considered to contain 300 micrograms of pantothenic acid. This means that the 
heef livers contained approximately 150 micrograms per gram of the dried tissue. 

From such calculations the data presented here can he augmented by values 
of approximate pantothenic acid content of meat. This is not a fully adequate 
procedure since the minimal protective level must necessarily he for a group of 
3ample3 of a particular tissue. The individual samples of the meats will un- 
doubtedly show some variation in their pantothenic acid content that will not he 
reflected in the calculations from the minimal protective level. Some indica- 
tion of this individual variation can he seen from several of the 3erie3 in which 
same levels of two tissues did not agree in their dermatitis protecting ability. 
This approximation is useful, nevertheless, in obtaining relative comparisons of 
potencies between tissues. In table XX such average figures are presented. As 
will be shown in the following section, the development of a pantothenic acid 
microbiological assay has enabled a further check on these assays; and since a 
larger number of samples could be assayed by the new procedure, we decided to 
review our results using the microbiological assay. 

Microbiologi cal Assay for Pa ntothenic Acid 

Although the chick assays furnished a qualitative estimate of the panto- 
thenic acid content of the meats, it is now possible to obtain a more quantita- 
tive measure of this substance in biological materials. A great many assays on 
a greater variety of samples can now be performed in less time and with less 
effort than was possible by the chick method. In addition to the greater con- 
venience, the accuracy is also improved. Many of the samples that were avail- 
able for the chick assays were retested by the microbiological procedure.' In 
order to make these assays more extensive, numerous samples have since been 
accumulated which can augment the data obtained by the previous biological method, 
The samples assayed by the bacterial method now total over 80. 

Method of Assay 

The microbiological a.ssays have been made by the bacteriological technique 
developed by Feeney and Strong (lh) . The method depends upon the growth of a 
pure culture of Lactobacillus casei e ( Lactobacillus helveticus ) on a media which 
is deficient in pantothenic acid but complete in the other essentials for opti- 
mum growth. The basal media contains NaOE-treated-peptone 0,5 per cent, sodium 
acetate 0.5 per cent, glucose 1 per cent, cystine 0.01 per cent, asparagine 
0.025 per cent, riboflavinl.O microgram per 10 cc . of media, and a yeast supple- 
ment equivalent to 50 .milligrams autolyzed yeast per 10 cc. media, and mineral 
salts 0.05 cc. each of solutions A and B described by Snell and Strong (5*0 for 
the riboflavin as3ay3. The important change in this media from the riboflavin 
test media consists in removing an far as possible the pantothenic acid which 
occurs in the yeast extract and in the peptone. The yeast supplement is made 
acid and nor i ted with two portions of activated, charcoal. The resulting water 



179 

white extract is neutralized with NaOH. The peptone is treated with NaOH to 
destroy any pantothenic acid which is present. The addition of asparagine to 
the media has improved the linear relationship "between growth of the bacteria 
and added pantothenic acid. 

The assays were set up so that each sample was tested at three levels and 
each of those levels in duplicate. In order to approximate the half optimum 
growth of the organism, it was necessary to dilute part of the sample extract 
to a convenient. volume so that the level of pantothenic acid was in the range 
0.0^- to 0.10 micrograms. Various levels of the diluted extract were used 
ranging from 0.5 ml. to 5.0 ml. In each series of assay tubes it was necessary 
to determine the standard curve of the response of the microorganisms to known 
amounts of crystalline calcium pantothenate. After t':e extracts of the samples, 
the crystalline standard solution, and the media were added to the test tube3, 
the volume was made to 10 ml., with distilled water. The tost tubes were 
stoppered with cotton plugs and autoclaved for 15 minutes. After cooling, the 
tubes were aseptically inoculated with a normal saline suspension of a freshly 
cultured inoculum of Lactobacillus casei e . which passed through one subculture 
in the liquid media. The tubes were incubated for three days at 37° C. and at 
the end of this time the acid produced ~oz r the growth of organisms in each tube 
was measured by titrating with N/lO IfeOH. By plotting the acid production, 
against pantothenic acid concentration, a standard reference curve was obtained 
which was then used for calculating the amount of pantothenic acid in each tube 
and from that the amount in the original. sample. These calculations have. been 
shown to be valid since within a certain range, the amount of acid produced is 
directly proportional to the concentration of pantothenic acid. When the titers 
were out of the range of greatest accuracy, the assays were repeated. In all 
cases the values obtained were the average of at least two different assays but 
more often three or four. 

The chick assays furnished an estimate of the approximate content of panto- 
thenic acid in the tissues which were used .in the preliminary runs of the micro- 
biological assay. One gram samples of liver, kidney, and heart were used, while 
5 to 7 3&. samples of the muscle and other tissues were used. In later deter- 
minations only 0.5 gram of sample was used which allowed greater dilution range 
aa well as the convenience of taking more suitable all quota. 

In order to check the. effect of the drying method on the pantothenic acid 
content of the meats, several samples were assayed in the fresh and in the dry 
state. It wa3 also necessary to determine the preferable method of preparing 
these samples for the bacterial assay since some indication was obtained by 
Rohrman et al (53) that in order to obtain maximum growth of the yeast prepara- 
tions, it wa3 necessary to free the pantothenic acid molecule from its natural 
linkages, probably proteins. Pennington et al (kj) found that it wa.s necesaary 
to autolyze the tiaaues before the method waa reproducible. The method of 
preparation of the sample followed for part of the preliminary assays was simply 
to weigh out the finely divided dried tissue and place it in a 125 cc. crlen- 
meyer flaak. After inaerting a cotton plug,- the flaak wa3 autoclaved for 20 
minutes at 120° C. and 15 pounds pressure. The cooled solution was then trans- 
ferred to a Varing mixer and the material further dispersed on the high speed 
knives. The sample wa,s made to a convenient volume. 



Variable results were obtained by this method of preparation of the samples. 
It was found that two different extracts of the same sample gave values that 
3howed only a fair comparison. It appeared necessary to obtain more consistent 
results if the assays were to be valid. Several meat samples were obtained from 
the neighborhood market and subjected to a number of different procedures in 



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order to determine the "best method of extraction. The samples were veal heart, 
pork loin, "beef round, veal liver, and pork liver. These were representative 
of "both high and low pantothenic acid containing tissues. Part of each sample 
was assayed in both the fresh and dry state, and portions of the fresh tissue 
were "both autolyzed and pepsin digested. After determining moisture content of 
the fresh tissue it was possible to calculate the loss of pantothenic acid which 
may have occurred in the drying procedure. All the samples used in the extraction 
+ rials were homogenized in the Potter-Elveh.iem homogenizer (U9). 

At the conclusion of these trials it was apparent that the drying procedure 
had no deleterious effect on the pantothenic acid content. The vitamin content 
of veal heart, pork loin, and beef round in the dry state was equal to that of 
the fresh weight when the pantothenic acid content was expressed in micrograms 
per gram of the dried tissue. The results of the various extraction procedures 
are summarized in Table XXI. There is an increase of both the autolyzed and 
pepsin digested tissues over the non hydrolyzed tissues, and the values of the 
dry pepsin digested meats compare very well with the figures for the fresh pepsin 
digested meats. It will be seen that there is excellent agreement in the pork 
liver sample with the exception of the homogenized fresh pepsin digested portion. 
This figure is evidently in error. There is good evidence then that pepsin 
digestion makes more pantothenic acid available to the microorganisms. It may 
also "be that some stimulating material is liberated by the pepsin digestion, . but 
several experiments have given evidence that the entire amount of pantothenic 
acid added can be completely recovered within experimental error. 

Further work in our laboratory has been done on the effect of other enzymatic 
preparations in liberating the pantothenic acid of animal tissues. Although the 
pepsin digestion of the meat samples apparently increased the pantothenic acid 
content of the tissues, digestion of the meat with an enzyme preparation called 
"clarase" increased the assay value slightly more. Rather complete liquefaction 
of the material took place during the digestion in the 37° C incubator. Since 
the clarase preparation was very high in phosphatases it was thought advisable 
to U3e a pancreatic extract which contains .more proteinases and similar enzymes 
which would completely hydrolyze the meat proteins, and thus free pantothenic 
acid from its natural linkages. Several trials with the enzyme preparations on 
crystalline pantothenic acid satisfied us that the enzymes had no effect on the 
amide linkage and that very good recoveries could be obtained. There appears to 
be a still further increase in pantothenic acid content after pancreatic diges- 
tion, and further trials are now in progress to determine whether stimulating 
substances are liberated which cause an increased fermentation of the organism, 
or whether there is an actual increase in the liberated pantothenic acid. 

It can be concluded from the work just cited that the state of division of 
the sample, the availability of the pantothenic acid to the organism, and the 
type of digestion of the tissue are of prime importance in the determination of 
the vitamin. In several of the digestion trials, enzymatic hydrolysis doubled 
the pantothenic acid content. The values for pantothenic acid which are deemed 
the most reliable at present are given in Table XXII. No attempt is made to 
present the complete data on the various types of extraction or the several 
repeat runs which were performed on a particular extract. The table thu3 gives 
the most acceptable figure at present for the pantothenic acid content of a 
particular tissue. 



Results of Microbiological Assay 

The livers of the various species again show the highest pantothenic acid 
content and thus corroborate the observations made in the chick experiments. 



182 



Sample 



TABLE XXII 

P anto theni c Acid Content of Animal Tl s a uea 

Determined "by Microbiological Assay 



Sample 


Treatment 


Number 


of Meat 


121 


Fried 


150 


Dried 


152 


Dried 


98 


Dried 


131 


Dried 


97 


Dried 


70 


Dried 


1-38 


Dried 


122 


Dried 


1^3 


Dried 


159 


Dried 


86 


Dried 



Micrograms of Pantothenic Acid 
Per Gram Per Gram 
Dry Weight Fresh Weight 



Beef Liver 



Veal Liver 



Pork Liver 



Lamb Liver 
Eeef Kidney 



Perk Kidney 



Beef Heart 



Veal Heart 
Pork Heart 



96 



Dried 



126 


Dried 


81 


Dried 


112 


Stewed 


137 


Dried 


83 


Dried 


62 


Dried 


133 


Dried 


54 


Dried 


lkj> 


Dried 


87 


Stewed 


155 


Dried 


ic4 


Dried 



98 

220 

186 
139 
251 

230 
190 

1^6 

125 
218 

192 
108 

184 

183 
162 
76 

170 
153 
123 

112 
26.5 
77.6 
23.7 

91 



^2.0 

60.5 
5 ] +.8 

73.3 

66.7 
50.5 
39.4 

39.4 

67.5 

53.7 
34.8 



3.4 



36.6 

36.7 
27.0 

34.2 

33.5 

26.6 

21.6 

5* 

14.8 

7.1 

30.5 

20.6 



183 



TABLE XXII (Continued) 



Sample 



Sample' 


Treatment 


Number 


of Meat 


130 


Tried 


76 


Dried 


109 


Steved 


38 


Stewed 


128 


Dried 


te 


Dried 


105 


Dried 


106 


Fried 


107 


Roasted 


lk6 


Dried 


iM 


Broiled 


148 


Fried 


151 


Dried 


157 


Dried 


154 


Dried 


95 . 


Dried 


32 


Dried 


139 


Dried 


l4l 


Fried . 


142 


Roasted 


52 


Fried 


124 


Dried 


36 


Dried 


37 


Broiled 


33 


Fried 


74 


Dried 


125 


Dried 


94 


Roasted 


93 


Fried 


89 


Dried 


156 


Dried 



Micrograms of Pantothenic Acid 
Per Gram Per Gram 
Di\, Weight Fresh Weight 



Beef Spleen 



Beef Round 



Fork Ham 



Pork Loin 



(Fre3:i Frozen) 



50 

42.6 
20 
19.6 

25 

24.8 
36.6 
12.1 

37.5 
23.2 

20.5 
16.7 

8.5 
16.9 
32.8 

45 

53 

28.1 

12.8 

10.7 

14.2 

75 

24 

14.9 

21.3 

30.8 

42.0 

10.5 

17.4 

36.I 

30.7 



10.3 



3.9 



6,6 



10.0 

8.0 

5.9 



2.1 

4.75 
8.1 

11.0 

14.6 

7.1 



17.7 

7.5 
3.1 
5.1 
8.7 
11.6 

2.7 
5.7 
12.0 
6.21 



181* 



TABLE XXII (Continued) 



Sample 



Smoked Ham 



Tender Made Ham 



Boiled Ham 



Veal Hindquarter 



Sample 


Treatment 


Number 


of Meat 


47 


Dried 


48 


Fried 


102 


Dried 


116 


Dried 


72 


Dried 


119 


Dried 


11? 


Dried 


115 


Dried 


101 


Dried 


129 


Dried 


^ 


Fried 


134 


Dried 


103 


Dried 



Lamb Leg, 
Beef Pancreas 

Beef Brain 

Beef Lung 

Beef Tongue 

Chicken, Light Muscle 

Chicken, Dark Muscle 
Filet of Cod 



Micrograms of Pantothenic Acid 
Per Gram Per Gram 
Dry Wei glit Fresh Weight 



80 



82 



Dried 



113 


Dried 


64 


Dried 


132 


Dried 


144 


Dried 


78 


Dried 


138 


Dried 



Dried 



6Q 


Dried 


65 


Dried 


66 


Dried 


35 


Dried 


36 


Dried 



30 
17.3 

33.3 
28.8 

13.3 
21.0 

17.5 

7 
21.5 

60 

31.0 
42.0 
53.0 

39.5 

98 
52 

76.7 
74.0 

47.4 
53.8 

38 

23.O 
23.4 
50.1 

14.0 

25 .4 



9.75 

8.1 

9.5 

3.5 

k.2 
7.0 
5.8 

2.k 
7.2 

15.0 

9.1 
14.2 

10.0 

25.6 
16.7 



16.6 
15.9 



11.8 

10.6 

5.6 

6.4 

12.9 

2.4 
7.1 



185 

Beef heart and spleen were next in order of activity and were considerably above 
"beef lung, pancreas, tongue, and brain. Beef muscle samples showed fairly con- 
sistent range of values, while the pork muscle samples showed a somewhat wider 
range. The veal muscle samples showed similar variation, while the two samples 
of light chicken were nearly identical. The dark muscle of the chicken contained 
more than twice that found in the light chicken muscle. 

The effect of household cooking and commercial processing on the panto- 
thenic acid content of the meats is readily brought out by examination of the 
table. There were several samples which could not be compared to the parallel 
untreated tissue, but a fair comparison can be made to other samples of similarly 
untreated tissues. The sample of fried beef liver showed a loss of pantothenic 
acid when compared to the untreated tissues. When a sample of beef round was 
fried there was appreciable loss of pantothenic acid. Similarly a sample of pork 
ham showed definite loss of the vitamin. It is apparent that different samples 
of meat will be affected differently by the cooking processes employed, since a 
sample of fried pork loin showed only a slight loss. 

The roasting procedure evidently did not cause much destruction of panto- 
thenic acid in the sample of roast round of beef, but when pork ham was roasted 
the loss was considerable. Stewed kidney, heart, and spleen showed a definite 
loss of pantothenic acid. 

Discussion of Pantothenic A cid Results 

A comparison between the approximate pantothenic acid content of the meat 
tissues obtained by the chick assay and the values taken from the microbiological 
assays shows very good agreement for most of the samples. The values obtained 
from the chick assays for liver, kidney, and most muscles fall in the range found 
by the microbiological assay. Those groups which were fed high levels of tissue 
showed the greatest variation from the microbiological assay. In general however, 
the values are in good agreement. The same order of potency in the meat3 was 
observed in both determinations. The liver, kidney, and heart showed the great- 
est content of pantothenic acid. The spleen, muscle, and other organ tissue 
followed the more active tissues. 

The results of the chick assays are in agreement with those of Jukes and 
Lepkovsky (25) who found that 2^ per cent meat scraps was only a fair source of 
the "filtrate factor". Juices (23) fed vacuum dried beef round at 25 and 50 per 
cent levels and concluded that it had a "low filtrate factor" content. 



The loss of pantothenic acid during cooking and commercial processing is 
undoubtedly explained in part by the solution of the vitamin in the water of the 
meat which is lost during the heating. The loss may also be attributed in part 
to the actual destruction of the vitamin by the heat. The stewed meats lost 
pantothenic acid into the cooking water. Although the water was not tested, there 
is evidence that pantothenic is leached from the meat into the cooking water. 
Approximately 50 per cent of the vitamin was lost by stewing kidney and heart. 
The loss of the vitamin in stewing spleen amounted to very nearly 60 per cent. 

The effect of roasting on the pantothenic acid was clearly shown in the 
samples, beef round sample 107 and pork ham sample 1^-2. The dried untreated 
tissue was assayed at the same time as the roasted tissue and all conditions of 
the assay were kept as constant as possible. There was no loss due to roasting 
of the beef round. The loss observed in roasting of the pork ham was nearly 62 
per cent. The differences observed in these samples is not readily explained 
since the treatment of the two samples was very similar. A marked loss of 



186 

pantothenic acid was also observed in the sample of roasted pork loin. The 
broiled pork sample showed a loss of 28 per cent while the broiled beef round 
showed only a 9 per cent loss. 

An interesting observation was made in the assay of the fried liver samples. 
As described in a preceding section of this chapter it appeared that better 
growth was obtained in the chicks receiving the 1 per cent fried liver than those 
which received 1 per cent of the untreated liver. There wa3 no apparent explana- 
tion for the finding in the chick assays , and unfortunately the same sample 
which showed the better growth after frying wa3 exhausted and so could not be 
determined by the microbiological test. Another sample of liver wa3 fried and 
this time the microbiological assay showed a pronounced loss in pantothenic acid 
potency when compared to the assay of several other untreated liver samples. 

At the present writing no definite information is available on the need of 
pantothenic acid by humans. There is no clinical evidence that man requires this 
vitamin other than preliminary experiments which indicate that patients subsist- 
ing on poor diets show increased retention of the vitamin. It would appear from 
experiments with rats, dogs, and pigs, that the need for this vitamin is approx- 
imately 5 to 10 times the amount of thiamin or riboflavin that is required. 
Until further work is done on this question no absolute figure can be given. 









187 



UTERATURE CITED 



1. 

2. 

3. 
k. 

5* 

6. 

7. 



9. 
10. 
11. 

12. 



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Bonner, J. and Oxtman, G. Growth of Plants in Vitro. Preliminary Experiments 
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Damashek, V. and Meyer son, P.G. Pigeon Dermatitis - A Vitamin 3 Deficiency 
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Dann, W.J. and Subbarow, Y, Differentiation of the Rat Dermatitis Factor and 
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Dimick, M.K. and Lepp, A. Relation of Pantothenic Acid to the Filtrate 
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Edgar, C.E. and Macrae, T.F. Water Soluble B Vitamins, Properties of 
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Elliott, A.M. The Influence of Pantothenic Acid on the Growth of Protozoa. 
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El Sadr, M.M., Hind, H.G., Macrae, T.F., Work, C.E., Lythgce,. B., and Todd, 
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Elvehjem, C.A. and Kbehn, Jr., C.J. Studies on Vitamin Bg (G). J. Biol. 
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Elvehjem, C.A. and Xoehn, Jr., C.J. The Won Identity of Vitamin Bg and 
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Elvehjem, C.A., Madden, R.J., Strong, F.M., and Woolley, D.W. The Isolation 
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13. Evans, W.C., Uandley, W.R.C., and Happold, F.C. Nutrition of Corynebacterium 
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188 

17. Gytfrgy, P., Poling, C.E., and Subbarow, Y. Experiments on the Ant I Dermati- 
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18. Hi tchi"ngs, G.H. and Subbarow, Y. The Rat Growth Factors of the Filtrate 
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19. Hoffer, M. and Reichstein, T. Activity of /3 -Alanine in Stimulating Growth 
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23. Jukes, T.H. Further Observations on the Assay, Distribution and Properties 
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25. Juices, T.H. and Lepkovsky, S. The Distribution of the "Fitrate Factor" (A 
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26. Kline, O.L., Keenan, J. A., Elvehjem, C.A., ,and Hart, E.B. The Use of the 
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27. Koehn, Jr., C.J., and Elvehjem, C.A. Studies on Vitamin B 2 (G) .and Its 
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23. Koehn, Jr., C.J., and Elvehjem, C.A. Further Studies on the Concentration 
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29. Koser, S.A., Finkle, R.D., Dorfman, A., Gordon, M.V., and Saunders, F. 
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30. Lease, J.G. and Parsons, H.T. The Relationship of Dermatitis in Chicks to 
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32. Lepkovsky, S. and Jukes, T.H. The Effect. of some Reagents on the "Filtrate 
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36, 



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Pantothenic Acid. Bicchem. J., _3_4, 1335 (19^0). ■■• 

Mc Burney, C.Hi, Bollen, W.B., and Williams, R.J. Pantothenic Acid and the 
Nodule Bacteria Legume Symbiosis. Proc. Natl. Acad. Soi., 21, 301 (1935); 
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McElroy, L.W. and Goss, E. Report on Four Members of the Vitamin B Complex 
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McElroy, L.W. and Goss, E. Synthesis of Members of the Vitamin B Complex in 
the Rumen of the Cow. J. Biol. Chem.., 133, Ixv (l9'+0). 

:. Pantothenic Acid and Growth of Streptococcus Hemolyticus . Brit, 
J. Exper. Path., 20, 330 (1939). 



37. 

33. Mcllwain 

39. 



McKlbbin, J.M., Madden, R.J., BlacK, S., and Elveh.jem, C.A. The Importance 
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kO. McKlbbin, J.M., Schaefer, A., and Elvehjem, C.A. '(Unpublished Data). 

kl. Mickelsen, 0., Waisraan, S.A., and Elvehjem, C.A. Inactivity of Nicotinic 
Acid in Chick Dermatitis. J. Biol. Chem., 124, 313 (1938). 

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4-3. Mueller, J.E. and KLotz, A.W. Pantothenic Acid as a Growth Factor for the 
Diphtheria Bacillus. J. Am. Chem. Soc., 60, 3086 (1938). 

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^5. Oleson, J.J., Woolley, D.W., and Elvehjem, C.A. Is Pantothenic Acid Essential 
for the Growth of Rats? Proc. Soc. Exp. Biol, and Med., 42, 151 (1939). 

46. Pelczar, M.J. and Porter, J.R. Pantothenic Acid and Nicotinic Acid as 
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47. Pennington, D., Snell, E.E. and Williams, R.J. An. Assay Method for Panto- 
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43. Phillips, P.E. and Engel, R.W. Some Eistopatho logical Observations on Chicks 
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49. Potter, V.R. and Elvehjem, C.A. A Modified Method for the Study of Tissue ... 
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51. Richards, O.W. The Stimulation of Yeast Proliferation by Pantothenic Acid. 
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190 

52. Ringrose, A.T., Norris, L.C, and Heuser, G.F. The Occurrence of a Pellagra 
Like Syndrome in Chicks. .' Poultry Sci., 10, 166 (1931). 

53. Rohrman, E., Bur get, G.E., and Williams, R.J. Pantothenic Acid Content of 
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191 

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CHAPTER XIV 
ADDITIONAL VITAMINS 

Choline _ _ 193 

Inositol _ _ 19k 

Anti -Grey Hair Vitamin ..... 195 

Biotin „ _ _ , 197 

Other Factors _ 197 

Literature Cited _ - _ - ....199 



L. 



CHAPTER XIV 



ADDITIONAL VITAMINS 



There exist several chemical substances other than those described in 
preceding chapters which are now considered as vitamins. In order to present 
a more complete summary of the vitamins it is necessary to mention several of 
these factors. Some of these newer vitamins are well known organic compounds 
and have only recently become recognized as biologically important entities. 
Among these chemical substances are choline, inositol, biotin, and para aminoben- 
zoic acid. The existence of these compounds in natural materials has been sus- 
pected but very little is known about the quantitative distribution of any one 
of the materials. It will be the purpose of the following paragraphs to. present 
a short discussion on the role of these compounds in nutrition and their occur- 
rence in animal tissues. 

Choline 



This compound has received much attention since the original observations by 
Sir H. Dale and by Professor 0. Loewi on the importance of this chemical as part 
of acetyl -choline, the chemical mediator for nerve impulse transmission. Several 
derivatives of choline have received attention by physiologists for their action 
upon the gastrointestinal tract and sympathetic nervous system. 

Choline is now considered as the sixth crystalline member of the vitamin B 
complex. It was shown by Best et al (5) in 1932 that choline prevented the develop- 
ment of fatty livers in rats on certain diets. Du Vigneaud et al (^6) demonstrated 
that choline was important in the relationship between homocystine and methionine 
and that choline was concerned in the ability of homocystine to replace methionine 
in the diet of the rat. In 1935 McHenry (30) reported that choline exerted some 
effect on the growth of young rats. The recent work of Griffith and Wade (13, 1*0 
and of Gytfrgy and Goldblatt (15) has given definite evidence that an enlargement 
and hemorrhagic degeneration of the kidneys occurs in rats on low choline diets. 
Sure (hk) has concluded that choline is an indispensable component of the diet 
for the growth and lactation of the rat. The hemorrhagic degeneration of the 
kidneys can be prevented by low levels of choline, but higher levels are necessary 
to prevent the fatty degeneration of the liver. Griffith (12) has given some 
indication that the amounts of cystine and methionine in the diet will influence 
the quantity of choline necessary. It was shown that the amount and nature of 
the fat wa3 also a consideration that determined the amount of choline necessary. 
Griffith (12, 13, 1*0 has concluded that no hemorrhagic degeneration occurs in 
young rats if a low choline diet contains more than 0.8 per cent of methionine. 
This worker relates this effect to the ability of methionine to furnish methyl 
groups for the synthesis of choline. If the ration contains less than 0.8 per 
cent methionine, the choline requirement will vary inversely with the dietary 
methionine and will be increased by the amount of cystine in the diet. Hegsted, 
Mills, Elvehjem, and Hart (19) have shown that choline is essential for the normal 
nutrition of chicks. Jukes (2*0 has reported that choline will prevent porosis 
or slipped tendon in turkeys on purified diets. Hegsted et al (19) have also 
found choline to be necessary for the prevention of perotic symptoms in the chick. 

Schaefer, McKibbin, and Elvehjem (39) have shown that choline is an essential 
constituent of the diet for young puppies. These workers used a purified diet 
containing sucrose, cottonseed oil, purified casein, and salts together with the 
known crystalline vitamins. After a definite plateau in the weight curve after 
several weeks on the diet without choline, the administration of this vitamin 
gave a marked response in growth. 



■193- 



■ 



194 

Very little ia known a"bout the distribution of choline in animal tissues. 
Fletcher, Best, and oolandt (8) have given some figures for the choline content 
of rat tissues using a "biological assay method, in which the isolated intestine 
of the rabbit was used as the test object for all of the assays. The total 
choline content of rat tissues has been determined by these workers, but values 
are also given for the choline content of various animal tissues. Ox liver con- 
tained 270 mg. per 100 grams of the fresh liver. Pancreas contained 230 mg., 
muscle 76 mg., and the fat of the ox contained between 0.5 and 2.6 mg. per 100 
grams of the tissue. The pig pancreas was also high in choline, having 280 mg. 
per 100 grams of tissue. The cured side bacon from the pig contained kk mg., 
while the fat from cooking the bacon contained 6 mg., but the lard contained 
1 mg. per 100 grams of each material. Jacobi, Baumann, and Meek (23) have re- 
cently published on a choline-reineckate precipitation reaction method for the 
determination of choline and have analyzed rat tissues by their method. The 
values of these latter workers check well with the method of Fletcher, Best, and 
Solandt (8). 

The few choline values available clearly demonstrate that animal tissues 
are a rich source of this compound, and that liver again is the tissue containing 
the highest amount of choline. Pancreas shows a surprisingly high amount of 
choline. 

Inositol 

An organic compound of .wide natural occurrence and biological significance 
is inositol. The earliest recognition of the importance of inositol was .made by 
the investigators working on the bios problem. A contribution by Eastcott (23) 
has been of prime importance in the bios problem since this worker reported the 
isolation and identification of bios I as identical with inactive inositol. How- 
ever, it was not until late in 19^0 that there was any indication that this sub- 
stance was required by the animal organism. Norris and Hauschildt (35) published 
a preliminary report that mice failed to grow on a synthetic diet supplemented 
with thiamin, pyridoxine, riboflavin, and nicotinic acid. A peculiar type of 
skin lesion developed that was different from the other dermatitis usually seen 
in rats. There was a loss of hair and a dandruff -like scale on the nude portion. 
It was shown that the factor was not found in a fullers earth filtrate. T //oolley 
(50) found similar results when he included the crystalline vitamins and choline, 
^-alanine, and a yeast extract. In addition to the symptoms obtained by the 
other workers there was observed a paralysis of the hind leg3 in some of the 
animals. The active substance which was relatively insoluble in alcohol and non 
dialyzable was found to be inositol (51). Pavcek and Baum (36) reported that 10 
mg. of i -inositol prevented the spectacled eye condition in rats maintained on a 
synthetic diet. Hegsted and coworkers (l8) have obtained some stimulation in 
growth of chicks on certain diets. 

The literature has scattered reports on the occurrence of inositol in animal 
tissues. Many of the values were obtained at. a time when there was no general 
agreement as to the best method of extraction of the substance from natural mater- 
ial. Several of the papers give only qualitative values since the purpose of the 
investigation was primarily one of obtaining information on the properties or 
stability of the compound. As early as 1908 Rosenberger (37) suggested a method 
for the determination of inositol in animal fluids and tissues but he soon pro- 
posed modifications of the original procedure (38). There existed great differ- 
ences in the proposed methods for the determination of inositol, but several 
workers reported the actual amount isolated from a particular tissue. Momose (32) 
obtained 0.32 grams from one kilogram of minced brain. Young (52) also proposed 
a method for the determination of the compound and recommended a variety of 



195 

extraction procedures. After deciding on the best extraction method he gave 1^9 
milligrams of inositol for 100 grams of ox train, 176 for sheep brain, 158 for 
sheep heart, 156 for dog heart, 27 for rabbit muscle, and less than 5 milli- 
grams per cent for ox muscle. A modification of Young's method has been offered 
by Gregory (11 ), -who found sheep heart to contain 80 milligrams per cent and dogs 
heart 1^0 milligrams per cent. The difficulty of extracting the inositol from 
animal tissues has been primarily one of preserving the compound from the autoly- 
tic processes that occur after the death of the animal. Winter (k-9) has pointed 
out these difficulties and has also cautioned against the use of alkaline extrac- 
tions. This author found 32-75 milligrams per cent in sheeps heart, 28-98 milli- 
grams in pig heart, and 55-7*+ milligrams in ox heart. 

These values are only an indication of the inositol content of animal tis- 
sues. Much more quantitative work is necessary to furnish' reliable values of 
this substance in meat and animal products. 

Anti-Grey Hair Vitamin 

There has been great interest, in this newest factor of the vitamin B complex 
as exemplified by many publications on the subject. Morgan, Cook, and Davison 
(33) were the first to note that the active substance which prevents and cures 
the greying is present in the filtrate fraction of the vitamin B2 complex. Lunde 
and Kringstad (28) have confirmed this observation and have concluded that the 
"rat growth" factor (vitamin Bg) and the "chick antidermatitis factor" (panto- 
thenic acid) are not identical with the anti-greying vitamin. Nielsen, Oleson, 
and Elvehjem (3*0 have obtained concentrates of the factor. 

The depigmentation of the black coat of fur ha3 been known to occur previous- 
ly in dietary deficiencies of Cu and of Zn. This condition is closely differen- 
tiated from the other dietary deficiency due to a lack of an organic factor. The 
appearance of the fur in the mineral deficiency is different from that of the 
organic factor deficiency. There is a more "rusty" appearance of the fur of the 
copper deficient rats, while the greying of the hair coat in the vitamin deficiency 
is more striking. It is possible that both the inorganic elements and the "anti- 
grey hair" vitamin are required to give normal growth and normal color of the 
hair. Free (9) has shown that the vitamin deficiency grey hair is not cured by 
additions of adequate amounts of copper, manganese, and iron. The grey hair pro- 
duced on the milk diet, due to the copper deficiency, is not aided by a rice bran 
extract. Unpublished results by Henderson, Mclntire, Waisman and Elvehjem (22) 
have shown that the greying of the fur of piebald rats maintained on a milk diet 
and supplemented with iron and manganese, was different in appearance than the 
greying observed on a synthetic vitamin deficient diet. There is more rustiness 
of the fur and spotted areas which is in contrast to the gradual greying observed 
on the latter diet. The administration of copper to the copper deficient rats 
resulted in a fine black fur in several weeks. Additions of pantothenic acid to 
the milk diet, together with other vitamins had no effect on the greying due to 
the copper deficiency. These workers have also reported that pantothenic acid is 
effective both in the cure and prevention of the greying of the fur in rats main- 
tained on a pantothenic acid free diet. 

A number of investigators have claimed that pantothenic acid is the grey 
hair factor (17, ^5). Other workers have contended (^4-8) that pantothenic acid 
has some action in the grey hair effect, but that it is not the only compound 
that is concerned. Further work on the pantothenic acid balance in the urine and 
feces of rats maintained on pantothenic acid low diets and with different levels 
of pantothenic acid may give some evidence on the mode of action of pantothenic 
acid and its relation to the greying. 



196 

From preliminary experiments designed to estimate the anti-grey hair vitamin 
content of animal tissues, it was apparent that meats must he considered as good 
sources of the factor. Experimenting on piebald rats, it wa3 found that 1 per 
cent of the liver tissue of pork and beef is able to furnish sufficient amounts 
of the vitamin. The rats received this supplement for 11 weeks ■ without showing 
any greying. A sample of veal liver was fed at 0.75 V Gr cent and the rats became 
very grey within 5 weeks., and even after 8-10 weeks there was no improvement. 
However, at a level of 1.5 per cent for the same periods the rats exhibited a 
markedly black and normal coat. 

The beef kidney that was fed at a 0.5 per cent level was unable to prevent 
the typical greying. Levels of the kidney at 1.0 per cent were just borderline 
since some greying occurred. Those animals which received 1,5 I> er cent beef 
kidney had shiny black coats and showed fine growth responses. Although we did 
not use growth as an index of the ant i -grey hair content of the meats some 
interesting observations were made. It was noted that when greying occurred on a 
supplement that allowed fair growth, there was more severe greying than when the 
supplement did not promote any growth. It was also found that if no pantothenic 
acid was in the ration, the greying was less severe than if small amounts of 
pantothenic acid, 10 to 20 micrograms, were administered per day. It appears 
then that small amounts of pantothenic acid will stimulate growth to a slight ex- 
tent over the basal and will thus allow faster growth of the fur which will come 
in grey at a faster rate than if no growth of the fur occurred. ' .. 

A number of muscular tissues were also fed to determine their anti-grey hair 
vitamin content. Pork ham gave good protection at levels of 6 per cent and above. 
Leg of veal and beef round were also able to give protection at these levels. A 
sample of smoked ham gave greying at an 8 per cent level as differentiated from a 
pork ham sample which protected at a 6 per cent level. Protection was also ob- 
tained with 6 per cent of beef spleen and, with k per cent of beef heart. 

Although the work on the grey hair factor is far from complete, it appears 
that the protective ability of the meats is in the order of the pantothenic acid 
content of these tissues. Liver and kidney at levels calculated to supply pro- 
tective levels of pantothenic acid appear to prevent the greying of the fur. 
These assays for, the grey hair factor content of meats must be considered as pre- 
liminary until the identity of the factor is well established by isolation and 
synthesis or until the role of pantothenic acid is clearly defined. Insufficient 
evidence is at hand on the interrelationship of the vitamins to speculate on the 
probable role of pantothenic acid in the prevention of the gradual greying of the 
black hair in rats maintained on certain diets. The small differences that exist 
in the rations used by different laboratories may account for the diverse results 
obtained, and not enough stress has been given to the influence of hereditary 
factors and to environmental conditions that prevail in the different laboratories. 

Ansbacher (2) has claimed that para aminobenzoic acid is the grey hair factor, 
and Martin and Ansbacher (29) have asserted that para aminobenzoic acid will cure 
the greying of the hair in mice which is caused by feeding hydro quinone. The 
results obtained in this laboratory (21) do not bear out the contentions of these 
workers, since on the sucrose, purified casein, corn oil and synthetic vitamin 
diet the piebald and black rats are not given any protection from the feeding of 
5 milligrams of para aminobenzoic acid per day. The feeding of hydroquinone to 
rats also has no effect on the graying of the fur. Although para aminobenzoic 
acid does seem to give some growth response when fed in addition to thiamin, 
riboflavin, pyridoxine, nicotinic acid, pantothenic, acid, and choline no anti- 
greying potency was observed in the particular strain of rats used in this labora- 
tory and fed the diet described. 



197 



Bio tin 



The outstanding work on this yeast nutrilite has "been done by Kflgl and his 
co-workers (25). Its empirical formula was claimed to "be CnHi30jN2 s - Tlie r °l e 
of biotin has been of importance in the growth of microorganisms and there is an 
extensive literature on the requirements of the many organisms. Only within re- 
cent months has it assumed importance in the normal metabolism of some of the 
higher animals. Gyflrgy and associates (l6) have shown that "vitamin H" is 
probably biotin and that it can prevent the typical egg white injury symptoms. 
Work by Hegsted et al (20) has given evidence that biotin or "vitamin H" (V?) is 
effective in preventing a dietary dermatitis in chicks which is similar to the 
symptoms observed in pantothenic acid deficiency. This dermatitis cured by 
biotin is not prevented by large amounts of crystalline calcium pantothenate. 

The distribution of biotin in animal tissues has been studied by K&gl and 
van Hasselt (26), Snell (hi), and Lampen, Bahler, and Peterson (27). Kogl and 
van Hasselt, using the yeast seedling growth method, found that the tissues from 
the dog, cow, and steer calf averaged approximately 0.007 micrograms per gram of 
fresh tissue. The brains of the cows and calf ranged from 0.00^ to 0.010 micro- 
grams per gram of fresh tissue. The suprarenal gland showed 0.007, thymus 0.00^8, 
pancreas O.OO56, lung 0.006, liver 0.007, spleen C.006, kidney 0.0132, and muscle 
of the hindquarter 0.0002 micrograms per gram of the fresh tissue. Some of the 
biotin values for the White Leghorn hen's tissues analyzed by K5q1 and van Hasselt 
are as follows: liver 0.066, spleen 0.0283, kidney 0.1025, heart 0.009^5, white 
breast muscle 0.00192, and dark leg muscle 0.00292 micrograms per gram of fresh 
tissue. According to the unpublished results of Snell (kl) a 20 fold increase 
in the biotin content of liver can be obtained on autolysis. The observations of 
Lampen et al (27) indicate that acid hydrolysis of the tissues will give large 
increases of biotin. These workers used the growth of a particular strain of 
Clos tri dium butylicum for their bioassays. Dried beef kidney contained 2.5 
micrograms per gram by acid hydrolysis which when expressed on the fresh basis 
would mean approximately 0.6 micrograms per gram. A defatted and water extracted 
kidney contained only slightly less than this figure which is good evidence for 
the belief that biotin is firmly "bound" in the liver and kidney and perhaps in 
most animal tissues. Another value by Lampen et al gave for pork kidney k,0 
micrograms per gram of dried tissue. Lean beef and lean pork contained approxi- 
mately one tenth that of the liver and kidney. 

Other Factors 

There is adequate evidence that rats grow well when they are placed on diets 
containing sucrose, purified casein, salts, corn oil and the six crystalline 
vitamins, thiamin, riboflavin, pantothenic acid, nicotinic acid and choline. The 
ability of rats to reproduce on this simple diet has been amply demonstrated by 
a number of laboratories, which have carried the animals through several genera- 
tions . 



It is thu3 becoming apparent that there are definite limitations in experi- 
ments with rats which are designed to elucidate further accessory food factors. 
Elvehjem and co-workers (6) have suggested that intestinal synthesis may play a 
part in the good growth observed in rats maintained on a sucrose diet. It was 
proposed that high levels of pantothenic acid stimulate the growth of certain 
microorganisms in the tract and these organisms in turn synthesize a number of 
factors which are utilized by the rat. Black and Elvehjem (h) have obtained 
evidence in favor of this hypothesis by feeding certain bacteriostatic agents 
which prevent the growth of organisms in the intestine. 



198 

Further work with dogs has given additional evidence for the existence of 
still other factors (kk) , When young puppies are placed on partially sjTithetic 
diet3 consisting of sucrose, purified casein, corn oil, salt mixture, and several 
of the crystalline vitamins, they will grow for a time but will soon show a 
plateau in their growth rate. The addition of 2 per cent liver extract will 
cause an immediate response in growth. If choline is added to the ration instead 
of liver extract, there is also a response in growth for several weeks but soon 
there will occur a second plateau in the weight curve. If a residue prepared . 
from liver extract after extraction with acid ether to remove the pantothenic 
acid or a preparation treated with alkali to destroy the pantothenic acid is 
added there is a still further growth response. There exists preliminary evi- 
dence that the dog requires in addition to the six crystalline factors an 
alkali stable factor and an alkali labile factor which is similar to but distinct 
from pantothenic acid. 

A further approach to the problem of differentiating newer factors of the 
vitamin B complex has been the use of chicks as the experimental animal. In con- 
trast to rats, chicks fail to grow on diets containing the crystalline vitamins, 
a source of carbohydrate, protein, fat, and minerals. From a large series of 
experiments in a number of laboratories, it has been shown that cartilage, liver 
extract, and yeast furnish a number of factors which produce good growth in chicks. 
The stimulatory effect of cartilage has now been shown to be due to a combination 
of chondroitin, glycine, and arginine. In the rations used ty Hegsted et al (l8) 
5 per cent of chondroitin, 1 per cent glycine, and 1 per cent arginine gave nearly 
normal growth when fed with liver extract, yeast, dextrin, soybean oil, and a 
salt mixture. Almquist and co-workers (l) demonstrated that glycine and chon- 
droitin were part of their "rice factor" or cartilage factor. One of the factors 
required ''oy chicks Is the so-called "factor U" first proposed by Stokstad and his 
co-workers (k2 } k$) . This factor is found in yeast and is readily extracted by 
methyl alcohol -water mixture. Schumacher, Norris and Heuser (ko) have separated 
two fractions from yeast by alcoholic precipitation and have called them frac- 
tions E and S. From preliminary reports it appears that there is some similarity 
between one of these factors and "factor U" . 

As newer accessory food factors are differentiated methods for their deter- 
mination will also become available thus furnishing the means by which their 
occurrence in many natural foodstuffs can be measured. The ability of meat to 
furnish many of the unidentified nutrilites for yeast, bacteria, and for some 
mammals is not unexpected 3ince liver and kidney are recognized as good sources 
of some of these unknown factors. 



199 



LITERATURE CITED 



1. Almquist, H.J., Stokstad, E.L.R., Mecchi, E. and Manning, P.D.V. Identifica- 
tion of the Rice Factor. J. Biol. Cham'., Ijk, 213 (19^0). 

2. Ansbacher, S. P. Aminobenzoic Acid, A Vitamin. Science, 93, l64 (19*H). 

3. Best, C.H., Hershey, J.M. and Huntsman, M.E. 'The Control of the Deposition 
of Liver Fat. Am. J. Physiol., 101, 7 (1932). 

4. Black, 3. and Elvehjem, C.A. ( Uhpubli shed Data) . 

5. Eastcott, E.V. Isolation and Identification of Bios I.j Its Absorption "by 
and Recovery from Yeast. Canadian Trans. Royal Society, Canada, (3) 22, 
III, 267 (1928). 

6. Elvehjem, C.A. Never members of the Vitamin B Complex. Given before 
Chicago Section of the American Chemical Society (19^0). 

7. Elvehjem, C.A., Koehn, Jr., C.J. and Oleson, J.J. A New Essential Dietary 
Factor. J. Biol. Chem., 115 , 707 (1936). 

8. Fletcher, J. P., Best, C.H. and Solandt, O.M. The Distribution of Choline. 
Biochem. J., 29, 2278 (1935). 

9. Free, A.H. Non Identity of Grey Hair Produced by Mineral Deficiency and 
Vitamin Deficiency. Eroc. Soc. Exp. Biol. & Med., hk, 371 (19^0). 

10. Frost, D.V. and Elvehjem, C.A. Factor W and its Relation to the Vitamin B 
Complex. J. Biol. Chem., 123, 23 (3.939). 

11. Gregory, R.A. A Modification of Young's Method for the Determination of 
Inositol in Animal Tissues. Biochem. J., 29, 2798 (1935). 

12. Griffith, W.H. Choline Metabolism. V. The Effect of Supplementary Choline, 
Methionine and Cystine and of Casein, Lactalbumin and Fibrin, Edestin, and 
Gelatin in Hemorrhagic Degeneration in Young Rats. J. Nutr., 21, 291 (19^1). 

13. Griffith, W.H. and Wade, N.J. Choline Metabolism. I. The Occurrence and 
Prevention cff Hemorrhagic Degeneration in Young Rats on a Low Choline Diet. 
J. Biol. Chem., 131, 567 (1939). 

Ik. Griffith, W.H. and Wade, N.J. Choline Metabolism. II. Interrelationship of 
Choline, Cystine, and Methionine in the Occurrence and Px^evention of Hemorrha- 
gic Degeneration in Young Rats. J. Biol. Chem., 132 , 627 (19^0). 

15. Gy6"rgy, P. and Goldblatt, H.J. Choline as a Member of the Vitamin B Complex. 
J. Exp. Med.,. 72, 1 (19^0). 

lo. Gy6*rgy, P., Melville, D.B., Burk, D. and du Vigneaud, V. The Possible 

Identity of Vitamin H with Biotin and Coenzyme R. Science, 91, 2^3 (194-0). 



Gytfrgy, P. and Poling, C.E. Pantothenic Acid and Nutritional Achromotrichia 
in Rats. Science, _92, 202 (19^0). 



17. 

18. Heg3ted, D.M., Briggs, G., Elvehjem, C.A. and Hart, E.B. (Unpublished Data) . 



200 

19. Hegsted, D.M., Mills, B.C., Elvehjem, C.A. and Hart, E.B. Choline in the 
Nutrition of Chicks. J. Biol. Chem. 138, ^59 (l9 ] +l). 

20. Hegsted, D.M. , Oleson, J.J., Mills, R.C., Elvehjem, C.A. and Hart, E.B. 
Studies on a Dermatitis in Chicks Distinct from Pantothenic Acid Deficiency, 
J. Nutri. 20, 599 (19*K)). 

21. Henderson, L.M., Black, S., Nielsen, E. and Elvehjem, C.A. Reported at the 
Chicago Meeting of the Federated Societies of Experimental Biology and 
Medicine. April 15, 19^1. 

22. Henderson, L.M., Mclntire, J.M., Waisman, H.A. and Elvehjem, C.A. Influence 
of a Mineralized Milk Diet on the Greying of the Fur Coat of Piebald Rats. 
(Unpublished Data). 

23. Jacobi, H., 3aumann, C.A.., Meek, W.J. The Choline Content of Rats on Various 
Choline-Free Diets/ J. Biol. Chem., 138, 571 (l9 ] +l). 

2k. Jukes, T.H. Prevention of Perosis by Choline. J. Biol. Chem., ljk, 789 (19^0) 

25. Kflgl, F. and Tennis, B. Uber das Bios Problem. Darstellung von Krystallier- 
tem Biotin aus Eigelb. Zeit. f. Physiol. Chem., 2^2, hj (1936). 

26. Kogl, F. and von Has3elt, W. Uber das Vorkommen von Biotin in Tierischen 
Organismus. Zeit. f. Physiol. Chem., 2^3, 189 (1936). 

27. Lamp en, 0., Bahler, G. and Peterson, W.H. (Unpublished Data) . 

28. Lunde, G. and Kringstad, H. Uber Verra'nderungen des Pelzes von Ratten Durch 
Mangel un Gewissen Faktoren des Vitamin B Complexes. II. Zeit. f. Physiol. 
Chem. 257, 201 (1939). 

29. Martin, G.J. and Ansbacher, S. Confirmatory Evidence of the Chromotrichial 
Activity of P. Aminobenzoic Acid. J. Biol. Chem., 133, kkl (19U1).. 

30. McHenry, E.W. An Effect of Choline upon the Weights of Young Rats. J. 
Physiol., 85, 3^3 (1935). 

31. McKibbin, J.M., Madden, R.J., Black, S. and Elvehjem, C.A. The Importance 
of Vitamin Bg and Factor W in Nutrition of the Dog. Am. J. Physiol., 128, 
102 (1939). 

32. Momose, G. The Inositol of Brain and Its Preparation. Biochem. J., 10, 120 
(1916). 

33. Morgan, A.F., Cook, B.3. and Davison, H.G. Vitamin B 2 Deficiencies as 
Affected by Dietary Carbohydrate. J. Nutr., 15, 27 (1938).' 

3k. Nielsen, E., Oleson, J.J. and Elvehjem, C.A. Fractionation of the Factor 
Preventing Nutritional Achromatrichia. J. Biol. Chem., 133, 637 (19^0). 

35. Norris, E.R. and Hauschildt, J. A New Factor of the Vitamin B Complex 
Required by the Albino Mouse. Science, 92, 3l6 U9 ] +0). 

36. Pavcek, P.L. and Baum, H.M. Inositol and Spectacled Eye in Rats. Science, 
93, 502 (19U1). 



201 



37. Rosenberger, F. Further Researches on Inosite. Zeit. f. Physiol. Chem., 
57, 464 (1908). 

33. Rosenberger, F. A Method for the Determination of Inosite in Animal Fluids 
and Tissues. Zeit. f. Physiol. Chem., 56, 373 (1908). 

39. Schaefer, A., McKibbin, J.M. and Elvehjem, C.A. Choline in Synthetic Diets 
for Dogs. Proc. Soc. Exp. Biol. & Med. (in Press). 

40. Schumacher, A.E., Heuser, G.F. and Norris, L.C. The Complex Nature of the 
Alcohol Precipitate Factor Required "by the Chick. J. Biol. Chem., 135, 313 
(194C). 

kl. Snell, E.E., as Quoted by Williams, R.J., in Biological Reviews, l6, 49 (1940). 

42. Stokstad, E.L.R. and Manning, P.D.Y. Evidence of a New Growth Factor Re- 
quired by Chicks. J. Biol. Chem., 125, 687 (1938). 

^3. Stokstad, E.L.R., Manning, P.D.Y. and Rogers, R.E. The Relation Between 
Factor U and Vitamin 3g. J. Biol. Chem., 132, 463 (I9h0) . 

44. Sure, B. The Essential Nature of Choline for Lactation and Growth of the 
Albino Rat. J, Nutr., 19, 71 (1940). 

45. Unna, K. and Sampson, W.L. Effect of Pantothenic Acid on Nutritional 
Achromotrichia. Proc. Soc. Exp. Biol. & Med. k$, 309 (1940). 

46. du Vigneaud, V., Chandler, J. P., Moyer, A.W. and Keppel, D.M. The Effect of 
Choline on the Ability of Homocystine to Replace Methionine in the Diet. J. 
Biol. Chem., 131, 57 (1939). 

47. du Vigneaud, V., Melville, D.B., Gy6"rgy, P. and Rose, C.S. On the Identity 
of Vitamin H with Biotin. Science, 92, 62 (1940). 

43. Williams, R.R. Inefficacy of Pantothenic Acid Against the Greying of Fur. 
Science, 92, 561 (1940). 

49. Winter, L.B. A Note on the Cardiac Inositol of the Sheep, Pig, and Ox. 
3iochem. J., 34, 249 (1940). 

50. Woolley, D.W. The Nature of the Anti -Alopecia Factor. Science, 92, 384 
(194C). 

51. Woolley, D.W. A New Dietary Essential for the Mouse. J. Biol. Chem., 136, 
113 (1940). 

52. Young, L. Determination of Inositol in Animal Tissues. Biochem. J., 28, 
1428, 1435 (1934). 



L. 



CHAPTER XV 

MEATS AS A SOURCE OF THE VITAMIN B COMPLEX 

Page 
Ability of a Single Food to Supply the B Vitamins 202 

Experimental _ _ _ _ _ 202 

Results _ _ 203 

Interrelationship of the Vitamins 20k 

Literature Cited _ 208 



CHAPTER XV 

MEATS AS A SOURCE OF THE VITAMIN B COMPLEX 

In the preceding chapters, we have discussed the distribution of the indi- 
vidual nutrients in meat. While these values will help greatly in formulating 
complete diets it is also important to consider the value of specific foods as 
sources of a certain group of factors. This is especially true in the case of 
the vitamin B complex, not "because there is any direct interrelationship "between 
the various members of the group "but because many of them at least take part in 
the same metabolic processes of the body. With the availability of at least six 
crystalline members of the vitamin B group, it is now possible to employ them in 
various combinations in both experimental and practical diets and compare the 
results obtained with those secured when natural foods are used. Although no 
single foodstuff is used as a source of all vitamins, it is important to know 
just what vitamins or group of vitamins will be supplied in that particular food 
if fairly large intakes of a single food will tend to produce any form of im- 
balance in the final diet. 

As part of the investigation on the nutritive value of meat, it was planned 
that tests be made which could furnish some information on the ability of a par- 
ticular tissue to supply the several members of the vitamin B complex. The 
ability of a tissue to supply the accessory food factors could in part bo pre- 
dicted by knowing the occurrence of thiamin, riboflavin, pyridoxine, pantothenic 
acid, nicotinic acid and choline content of that tissue. However, this was only 
partially satisfactory, since it was the purpose of this particular test to com- 
pare the effectiveness of a tissue in promoting growth and preventing nutritional 
symptoms not due only to the known members, but to the unknown food factors as 
well. Since only the potency of the known vitamins was previously determined, 
this test would furnish additional information on the content of other unidenti- 
fied vitamins . 

Several experimental approaches were possible for studying the ability of 
meat to supply the various members of the vitamin B group, but it was believed 
that the test would have more value if a practical ration were employed. The 
ration finally decided upon was the modified Goldberger ration. While this 
ration had been used successfully for the estimation of nicotinic acid with 
"black tongue" dogs, several laboratories had shown that rats will grow on the 
corn, casein, and fat diets. The work of Margolis, Margolis, and Smith (8) and 
of Helmer and Fouts (5) had given indications that this diet was low in ribo- 
flavin and in thiamin. Margolis, Margolis and Smith did their work with dogs 
while Helmer and Fouts used rats in their studies. Since there now exists an 
abundance of evidence that the chick requires many more factors preformed in the 
diet than does the rat, it was decided that the chick would be best for these 
experiments. By the use of this animal and the modified Goldberger diet it was 
possible to get an approximation of both the unknown and the known vitamins in 
animal tissues. 



Experimental 

The basal Goldberger diet used in this study was the same modification of 
the ration which was fed to dog3 in an earlier study by Waisman et al (15) and 
was also described in chapter XI. Day-old White Leghorn chicks were also used 
in this study as in our previous chick experiments, and the usual care was taken 
to supply additional A and D by a high potency fish oil concentrate. The birds 
were weighed weekly and carefully observed daily for any symptoms which might 
develop. The water solutions of the vitamins supplemented to the basal ration 
were dried on the ration. 

-202- 



203 

The levels of meat to "be fed were determined largely "by previous experiments 
In chick antidermatitis studies by Waisman.et al. Beef kidney had "been shown to 
"be equal to liver in its content of most of the vitamins and was thus fed at 
levels of 1, 2, 3, and 6 per cent. Beef kidney was used in all the trials involv- 
ing this tissue. The pork liver wa3 fed at 1 and 2 per cent levels, lamb liver 
at 1 and 2 per cent, and veal liver at 2 and h per cent. The beef spleen was fed 
at 5, 10> and 20 per cent levels. Muscular tissues were fed at several higher 
levels. Pork muscle was tried at 5, 10, 20, and 30 per cent, while veal muscle 
and beef muscle were fed at 10, 20, and 30 per cent respectively. 

In order to compare the growth obtained on the various levels of meats, 
2 per cent of liver extract and 5 per cent of brewers yeast were fed to the chicks 
maintained on the modified Goldberger diet. The group receiving the 2 per cent 
of the liver extract served as a control group since this fraction of liver is 
known to contain many of the water soluble vitamins. The 5 P er cent brewers 
yeast supplement was used as another positive control for the reason that this 
supplement contained the water soluble factors as well as the water insoluble or 
"residue factors". 

Result s 

Previous experiments by Waisman and Elvehjem (1*0 had given evidence that 
the modified Goldberger diet was unable to support good growth in chicks due to 
several outstanding deficiencies. Our data indicated that riboflavin was a 
limiting factor together with factor U and perhaps pantothenic acid and vitamin 
B/-. Thiamin exerted no effect when added to the basal diet, and nicotinic acid 
gave variable responses when fed at levels between 5 and 50 mg. per 100 grams of 
ration. In several groups there was a definite response to the added nicotinic 
acid, especially when other limiting vitamins were supplied in the diet. Vita- 
min Bg alone did not increase growth over the basal, but when added to a liver 
fraction which furnished additional factors, a definite increase in weight was 
observed. The basal ration was also low in pantothenic acid as indicated by the 
dermatitis shown in the birds of several groups and by the growth response when 
pantothenic acid was added to the diet. The effect of pantothenic acid addition 
was more striking when it was given with several of the other vitamins. This was 
undoubtedly due to the increased stimulation to growth by the other factors, and 
while the pantothenic acid in the corn supplied sufficient vitamin for chicks on 
the basal diet, the increased growth on the supplemented ration also increased 
the demand for pantothenic acid. 

Factor U also appeared to be present in borderline amounts in the basal 
ration. It had been shown by Stokstad and Manning (12) that 5 per cent of yeast 
is sufficient to furnish adequate factor U to the chicks. This no doubt accounts 
for the increased growth produced by the yeast over that obtained when liver 
extract was used as the supplement. In the chicks receiving the combination of 
several vitamins, growth was never optimum until a concentrate of factor U made 
from an adsorbate of yeast was used. The effect of factor U was demonstrated 
by adding the factor U concentrate to a number of combinations of the other 
vitamins already in the diet. It was also shown that additional factors, as yet 
unidentified, were required for optimum growth of chicks. 

The results of supplementing the Goldberger diet with various levels of 
animal tissues are given in table XXIII. A comparison of the weight gains on 
the various levels of meat with the gain on 2 per cent of liver extract and 5 per 
cent of yeast furnishes additional information on the ability of a particular 
tissue to -supply the various vitamins. Liver extract at a level of 2 per cent 



204 

in the diet produced a significant growth response "but did not give an optimum 
response. It will be seen that 1 per cent of liver or kidney gives protection 
from dermatitis and also allows some growth. With higher levels of liver or 
kidney there is obtained excellent growth and complete protection from dermatitis 
and leg unsteadiness seen in the basal chicks. The weight of the chicks receiv- 
ing the 3 per cent kidney and h per cent liver compare very favorably with the 
average weight of the chicks receiving the 5 per cent of yeast. 

Beef spleen evidently did not supply an adequate amount of the factors at 
the 5 per cent level as evidenced by the poor growth. Although there were no 
symptoms observed in the chicks at this level, an increase in the percentage of 
spleen in the diet gave markedly better growth. The muscle meats of pork, veal, 
or beef were apparently less effective at the lower levels as seen by the symp- 
toms of curled toes, borderline dermatitis around the eyes and beak, as well as 
a definite unsteadiness in some of the groups. With levels of meat above 20 per 
cent there was observed less severe symptoms and when 30 per cent of the tissues 
were fed no symptoms were observed in any of the chicks. When increasing levels 
of meat were fed, there was an accompanying increase in weight of the birds, but 
the highest level of muscle fed did not give the optimum response as judged by 
the growth obtained with yeast, kidney, or liver. 

In some of the groups there was seen an occasional case of slipped tendon. 
The birds which showed the leg deformity were usually those which grew very well 
within the experimental period. No explanation of these afflicted chicks can be 
given, but the most probable explanation is that hereditary causes, together with 
abnormally fast growth and borderline levels of choline in the ration resulted 
in a perotic chick. The number of chicks so afflicted was too few to have any 
significance. 

The most striking symptoms that were observed were those due to riboflavin 
and pantothenic acid deficiencies. The riboflavin deficiency was observed in 
those groups receiving the lower levels of the muscle meat, while the pantothenic 
acid deficiency symptoms were seen In the very low levels of the muscle tissue. 

Interrelationship of the Vitamins 

The results given above are partly the basis for several interesting specu- 
lations and observations. The groups which showed some dermatitis around the 
beak, eyes, and legs were those which were receiving some meat supplement. The 
levels of the meat that were fed did not supply sufficient pantothenic acid to 
give complete protection from the symptoms. It must be pointed out, however, 
that groups which received only the basal ration showed no symptoms of panto- 
thenic acid deficiency. The stimulation furnished by the tissue supplement indi- 
cated that several factors were furnished in the tissue and the increased growth 
that resulted evidently increased the need for pantothenic acid during the period 
of rapid growth which could not be supplied by the ration. No symptoms were ob- 
served in the groups receiving liver or kidney, two tissues which furnished 
additional amount of pantothenic acid to the ration. It is apparent then that 
certain tissues such as liver and kidney supply a well balanced mixture of vita- 
mins while muscle tissue although rich in vitamin B5 and nicotinic acid is some- 
what low in pantothenic acid. 



There have been numerous reports on the effect of varying the constituents 
of the diet on the vitamin requirement. Several studies have shown that the 
increase of fat in a thiamin-low ration lowered the thiamin necessary to prevent 
polyneuritis in the rat. This finding has been well established. Some work has 
also been done on the effect of increasing the protein or decreasing the protein 



205 



Supplements of 



TABLE XXIII 



Meat to the Goldberger Diet 



Modified Goldberger Sample Ave. wt. 
ration plus ■ number 5 "weeks 



Symptomatology 



None 

2$ liver extract 
5$ yeast 
1$ beef kidney 
1$ pork liver 
1$ lamb liver 
2$ beef kidney 
2% pork liver 
2$> lamb liver 
2$ veal liver 
14 " " 
5$ beef spleen 
3$ beef kidney 
5$ pork ham 
670 beef kidney 
lO/o beef spleen 
10$ pork ham 
10$ leg of veal 
20$ beef spleen 
20$ leg of veal 
20$ pork ham 
20$ leg of veal 
20$ beef muscle 
30$ pork ham 
30$ leg of veal 
30$ beef muscle 



126 
122 

96 

126 

122 

96 

70 

70 

130 

126 

32 

126 

130 

32 

103 

123 

103 

composite 

129 

128 

composite 

• 129 

,128 



Grams 
62 

170 

250 

190 

131 

119 
194 

209 
180 
236 
280 

99 

263 

88 

255 
180 

93 
io3 
177 
212 
202 
199 
165 

131 
1U7 
178 



Poor feathering - unsteady 
None 



Some dermatitis and unsteadiness 
None 

Curled toes - one bird 
Some leg weakness 
One leg unsteadiness 
Borderline riboflavin level 

None 






206 

on thiamin low diets, but further investigation on this problem is necessary. 
There has been observed a marked influence of fat on the riboflavin low diet (7). 
Fat appears to increase the riboflavin requirement of rats, and it also aggra- 
vates the typical denuding and scaliness of the skin in rats maintained on a 
dextrin diet. A high stepping paralysis has been observed in these animals. The 
effect of high protein on the riboflavin requirement has been negligible, yet the 
observation has been made that levels of protein ranging from 28 to 36 per cent 
definitely produce a typical vitamin Bg acrodynia not seen when l8 per cent 
casein is fed (h) . In this instance, strangely enough, the amount of pyridoxine 
necessary for normal growth remains the same. 

Some work has been done on the effect of abnormal metabolism on the inter- 
relationship and requirement of the vitamins by Sure and his associates (13) who 
have studied the thiamin and riboflavin requirement of animals receiving thyroxin. 
Since the rate of metabolic processes is increased, it appeared logical that the 
thiamin and riboflavin requirement would be increased. It has been established 
that injections of thyroxin also caused a decrease in the muscle and blood co zy- 
mase of rat3 maintained on a complete diet (6). When nicotinic acid was fed to 
these rats, the co zymase content of the tissues was brought back to a normal 
level. The vitamin A and vitamin D requirement has also been increased when 
thyroxin was injected according to several investigators. These are only several 
experiments which have been done on the effect of thyroxin administration. It 
is understandable that in the increased rate of metabolism, the need for the 
vitamins would also be increased in order to aid the greater rate of chemical 
reactions . 

The role of vitamins in a number of investigations not directly concerned 
with the accessory food factors has been apparent from several reports. One of 
these la the determination of the biological value of a protein by the usually 
accepted methods. The determination of the biological value of a protein has in 
the past been beset by the disadvantage that the various protein supplements 
differed in their vitamin content and when the determination of biological value 
was made it often was affected by the vitamins carried by the protein. With this 
fact in mind, Basu and Gupta (l) first autoclaved their casein and determined 
the loss of biological value then tested the effect of omitting all the members 
of the B complex or adding the individual vitamins in the separate trials. In 
one group, the yeast concentrate, marmite, was added, and in another group the 
marmite was withheld to determine the effect of the vitamins on the biological 
value. In the short period experiments of Basu and Gupta (l) it was found that 
omitting the vitamins from the diet always increased the biological value of the 
casein. The reason offered by these workers for this observation was that in the 
absence of the vitamin the metabolism and oxidative processes were reduced and 
thus caused a diminished elimination of nitrogen in the urine and therefore a 
greater retention thus indicating a better utilization of the protein. Although 
these experiments can be criticized in that only the labile vitamins were studied, 
the data point out the effect of the vitamins on the estimation of the biological 
va.lue of certain "oroteins. 



Perhaps the greatest possibility for studying the interrelationship of the 
vitamins lies in the use of purely synthetic diets. Although this work is 
limited to the known factors required by the rat, nevertheless certain unidenti- 
fied factors can also be studied. The work of Black et al (3) illustrates the 
use of the partially synthetic diet in the study of the effect of one vitamin or 
group of vitamins upon another. Black et al (2) found that high levels of panto- 
thenic acid replaced, in a large measure, the so called "factor W" supplied by a 
liver fraction. The addition of small amounts of liver preparations to a 
synthetic diet decreases the amount of pantothenic acid "necessary" for optimum 



20' 



growth. Since the liver preparation furnishes no appreciable quantity of panto- 
thenic acid, evidence is thereby presented to show that a factor in the liver 
can in some way "spare" the pantothenic acid requirement. 

■An approach to the problem which has a3 yet not been investigated is the use 
of diets containing quantities of vitamins in different combinations. It might 
be possible to get at the question of the increased or decreased need of a vita- 
min when other vitamins are supplied in suboptimal amounts. It would also be 
interesting to determine whether the addition of high levels of pantothenic acid 
to an otherwise complete ration or even to a riboflavin deficient diet would have 
any demonstrable effect on the growth or occurrence of symptoms. Although we 
usually think of a vitamin acting together with other vitamins to give the 
necessary normal chain of chemical reactions, there is no reason to assume that 
an excess of a particular vitamin will do no harm, nor conversely is there any 
justification for the belief that the need for a vitamin remains the same if a 
suboptimal amount of another vitamin is given. Also, nothing is known about the 
need for a particular factor if another vitamin is left out. 

Morgan (9) has asserted that some danger exists in administering only two 
or three vitamins to a dog suffering from a multiple deficiency. She presented 
evidence that more severe symptoms developed when only a few vitamins were given 
to dogs suffering from multiple deficiencies than when none were given. The 
intimation of this worker's report was that care must be taken in the administra- 
tion of only a few vitamins to individuals who are really suffering from a lack. 
of many vitamins since more severe symptoms may develop. This view can be criti- 
cized by the consideration that each vitamin has a definite function to perform 
and that no other vitamin can be replaced by another vitamin. The results of 
these workers are also not surprising 3ince it is well known that specific de- 
ficiencies can be produced only when fairly adequate quantities of other factors 
are furnished. If the animal does not grow the symptoms are usually mild or 
absent. A ration which increases growth or stimulates metabolism usually aggra- 
vates the condition related to the primary deficiency in the ration. 



In this connection an interesting observation has been made by a number of 
clinical investigators who were interested in the problem of pellagra and accom- 
panjang nutritional deficiencies (10, 11). It was observed that when nicotinic 
acid was given to patients with a very poor nutritional history, the severe 
pellagra which exemplified itself in the dermatitis of the hands, face, and legs 
would clear up; but there would then arise the symptoms of riboflavin deficiency. 
It was evident that the patient having a poor nutritional deficiency first showed 
the symptoms of the most pressing deficiency, and after it was cured the secondary 
deficiencies became more apparent. The typical fissures at the corner of the 
mouth appeared in a patient which was receiving nicotinic acid therapy, and it 
was then apparent that the patient was also suffering from a riboflavin deficiency. 
It has been said that the administration of one vitamin to a patient of marked 
nutritional deficiency exacerbated the symptoms due to the other deficiencies'. 
Although this is the apparent explanation it may also be that the more striking 
symptoms of the first deficiency covered the less severe symptoms of concurrent 
deficiencies. The use of milk and meat In the diets of these people suffering 
from multiple deficiencies is definitely indicated by virtue of the high content 
of the various members of the vitamin B complex. 

This chapter of the book has attempted to direct attention to the ability of 
meat to supply the factors of the vitamin B group and the need for further knowl- 
edge in knowing the vitamin content of other groups of foods. The authors have 
also tried to emphasize the importance of the interrelationship of the vitamins 
and the opportunities available in this field of study. With a greater apprecia- 
tion of the occurrence of the vitamins in the individual groups of foods it will 
be possible to form a more complete pattern of the well balanced diet. 



208 
LITERATURE CITED 

1. Basu, K.P. and Gupta, K. The Role of Vitamins and Calcium in the Diet in 
the Utilisation of Proteins. J. Ind. Chem. Soc, 16, kk9 (1939-^0). 

2. Black, S. and Elvehjem, C.A. (Unpublished Data). 

3. Black, S., McKlbbin, J.M. and Elvehjem, C.A. Proc. Soc. Exp. Biol. & Med., 
(In Press) (19^1). 

k. Conger, T.W. and Elvehjem, C.A. The Biological Estimation of Pyridoxine 
(Vitamin Bg). J. Biol. Chem., 158, 555 (19^1). 

5. Helmer, O.M. and Fouts, P.J. Multiple Nature of the Deficiency of Black 
Tongue Producing Diets as Shown "by Studies with Rats. J. Nutr., l6, 271 
(1938). 

6. Katzenellenbogen, E., Axelrod, A.E. and Elvehjem, C.A. (Unpublished Data) 
(19^0). 



7. Manner ing, G.J., Lipton, M.A. and Elvehjem, C.A. Relation of Dietary Fat 

to the Riboflavin Requirement of Growing Rats. Proc. Soc. Exp. Biol. & Med., 
k6 t 100 (19^1). 

8. Margolis, L.H., Mar go lis, G. and Smith, S.G. Secondary Deficiency of Vitamin 
B 1 and Riboflavin in the Black Tongue Producing Diet. J. Nutr., 17, 63 (1939). 

9. Morgan, A.F. The Effect of Imbalance in the "Filtrate Fraction" of the 
Vitamin B Complex in Dogs. Science, 93 , 26l (19^1). 

10. Sebrell, W.H. Vitamins in Relation to the Prevention and Treatment of 
Pellagra. J. Am. Med. Assoc, 110 . 1665 (1938). 

11. Spies, T.D., Bean, W.B. and Ashe, W.F. Recent Advances in the Treatment of 
Pellagra and Associated Deficiencies. Ann. Int. Med., 12, 1830 (1939). 

12. Stokstad, E.L.R. and Manning, P.D.V. Evidence of a New Growth Factor Required 
by Chicks. J. Biol. Chem., 125, 687 (1938). 

13. Sure, B. and Buchanan, K.S. Antithyro genie Action of Crystalline Vitamin B-j_. 
J. Nutr., 13, 513 (1937). 

Ik. Waisman, H.A. and Elvehjem, C.A. Multiple Deficiencies in the Modified Gold- 
berger Diet as Demonstrated with Chicks. J. Nutr., 20, 519 (19^0). 

15. Waisman, H.A., Mickelsen, 0., McKibbin, J.M. and Elvehjem, C.A. Nicotinic 
Acid Potency of Food Materials and Certain Chemical Compounds. J. Nutr., 
12, ^83 (191»0). 



CHAPTER XVI 
SUMMARY 



CHAPTER XVI 

SUMMARY 

The vitamin A content of muscular tissue is not of great significance. 
The liver of "beef, veal, pork, and sheep are good sources of the vitamin. It 
compares very favorably with the liver of various fish. No other mammalian 
tissue can compare with the liver in its vitamin A content. 

The vitamin D content of most animal tissues is indeed very low. Liver 
again furnishes the greatest amount of this vitamin of any of the tissues from 
any species. 

Vitamin C is distributed fairly evenly in most tissues, the greatest amount 
"being found in the adrenals. Liver, muscle, "brain, and kidney contain fair 
amounts of vitamin C. 

At present there is insufficient knowledge available on the distribution 
of vitamin E and vitamin K in animal tissues. 

Thiamin occurs in pork muscle to a greater extent than in any other tissue, 
and in general the muscular tissue is superior to organ tissue. Liver, however, 
does contain somewhat larger amounts of thiamin than the other organs. 

Riboflavin occurs in liver and kidney to the greatest extent and these tis- 
sues are the richest natural sources of riboflavin. Heart, spleen, pancreas, and 
lung contain low amounts. Muscular tissue from the several species contains the 
same amount of riboflavin. 

The nicotinic acid content of liver and kidney is again greater than in the 
other tissues. Muscle contains approximately half that of liver and kidney, but 
the muscle of pork, veal, and beef show a higher nicotinic acid content than 
tissues such as pancreas, lung, and spleen. The values given have been determined 
both by chemical analyses and "by bioassay procedures. 

The increasing importance of pantothenic acid in nutrition makes it advisable 
that accurate information be gathered on the distribution of this new factor in 
foodstuffs. The investigations using both chick assays and microbiological 
methods have shown that liver and kidney again contain abundant quantities of 
this vitamin. Beef brain, beef heart, and pork heart contain good amounts of the 
vitamin. All of the other tissues contain fairly high amounts of this food 
factor. In general no tissue of any of the animals studied was devoid of panto- 
thenic acid. 



Pyridoxine occurs in muscular tissue to an extent equal to that of liver. 
Only beef liver contained more pyridoxine than did the muscular tissue of beef or 
pork. Most samples of the muscle tissues contained amounts of pyridoxine which 
were in the range of the liver samples assayed or slightly higher. 

Preliminary bioassays performed on animal tissues give adequate demonstration 
that meat and meat products are good sources of the more recently recognized 
factors such as biotin, inositol, choline, and the anti-grey hair vitamin. Liver, 
kidney, muscle, and spleen contain good amounts of these newer factors. 

The tables listing the various vitamins which occur in meat can be considered 
as reliable since only those values were chosen which were obtained by comparison 
to crystalline standards. This is especially true for the members of the vitamin 
B group. The values for the vitamin A, vitamin D, and vitamin C content of animal 

-209- 



210 

tissues summarized from the available literature have also been limited to those 
experiments which have employed the crystalline vitamin as the standard or which 
were converted by means of a reliable conversion figure. 



TABLE XXIV 

Vitamin B Complex of Animal Tissues 

Ribo- Nicotinic Panto- Choline 

Thiamin flavin acid thenic Pyridcxine mg./lOO 

gm. 
fresh 



Animal 
tissue 


Mcg./gm. 
fresh 


.mcg./gm. 
fresh 


mg./lOO gm. 
fresh 


acid meg./ 
gm. fresh 


meg./ 
fresh 


Brain 
Beef 


2.5 


2.6 


6.0 


16.3 


1.5 


Heart 
Beef 


6.8 


8.8 


7.0 


18.0 


2.1+ 


Pork 


6.1 


11.2 


6.5 


20.6 


3.5 


Kidney 
Beef 


2.7 


20.5 


10.0 


37.0 


k.H 


Pork 


5.2 


19.6 


10.0 


31.0 


5.5 


Liver 
Beef 


3.8 


30.0 


17.5 


63.O • 


7.3 


Lamb 


k.l 


26.6 


15.0 


53 . h 


3.7 


Pork 


5.2 


27.0 


19.0 


50.0 


3-3 


Veal 


5.2 


33.0 


■ 16.5 


53.0 


3.0 


Lung 
Beef 


2.0 


h.9 


6.2 


11.8 


0.7 


Muscle 
Beef 


2.3 


2.6 


7.5 


6.0 


k.O 


Lamb 


3.0 


3.2 


8.0 


10.0 


3.0 


Pork ham 


15.2 


2.U 


8.0 


12.5 


6.1 


Veal hind- 
quarter 


3.5 


2.9 


8.0 


1^.5 


k.2 


Pancreas 
Beef 


3.2 


5.3 


5.0 


21.1 


2.0 


Spleen 
Beef 


1.6 


^.5 


7.5 


11.0 


1.2 


Tongue 
Beef 


2.8 


2.2 


6.0 


10.6 


1.2 


Poultry 
Light 


1.6 


0.8 


7.2 


6.0 


_ __ 


Dark 


2.k 


2.6 


6.5 


12.0 


2.0 



270 



76 



230 



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